Frequently Asked Questions
Here, you can browse and search for frequently asked questions, including customer inquiries about our catalog products, Q&A about structural biology-related experiments, and answers to general questions to make your ordering process smoother and more effective. If you have additional questions, please contact us by phone or email and our customer service representatives will get back to you within three business days.
- General
- Catalog Product
- Protein Technology
- Crystallography
- Cryo-EM
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Do you have promotions for bulk orders?
We need to evaluate this request further with specific information. You can describe your specific needs in the inquiry form, or call us directly.
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May I know the contact information of your distributor?
You can find contact information for all of our distributors here: Distributors.
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How should I use my coupon?
You can get the coupon information on the promotional page, generally speaking, please paste the code in the "Project Description" column of the form when making an inquiry or purchase.
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How to contact us?
Creative Biostructure offers 24/7 customer service. You can call us directly, send us an email to , or leave us a message on online chat tool and we will get back to you within 24 hours.
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When will you contact me after I submit the online inquiry form?
Normally, our technical support manager will contact you within 24 hours.
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Can I get a discount on the price?
Creative Biostructure runs promotions on holidays. Follow our website for the latest promotions. For specific promotions, please contact our customer service staff. Thank you for your inquiry, and we look forward to your order.
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How to ensure that the product reaches the customer safely and quickly?
We will put them in ice packs or use another packaging according to the storage requirements of the product to ensure safety and stability during transportation.
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How can I place an order?
You can place your order in four ways: through the inquiry form at the bottom of the pages, online chat tool, phone, and by email. Either way, we will confirm your order with you via the email provided.
Exosome Products
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Do quiescent cells secrete exosomes?
Yes, quiescent cells are known to secrete exosomes. Exosomes are small vesicles that are released by various types of cells, including both quiescent (non-dividing) and actively dividing cells. These vesicles are involved in cell-to-cell communication, as they carry a cargo of proteins, lipids, and nucleic acids that can be transferred to recipient cells.
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What strategies can be employed to standardize mRNA expression within exosomes?
You can utilize miRNA genes like miRNA16, miRNA26a, miRNA221, miRNA22, miRNA181a, miRNA181c, miRNA103, miRNA191, let7d, as well as small RNAs (5SrRNA and U6snRNA) that are normally expressed in exosomes.
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Do you offer items designed for conducting studies on tracking extracellular vesicles?
Creative Biostructure offers highly purified fluorescent exosome standards for tracking EVs in fluorescence microscopy experiments. These fluorescent exosomes are labeled with a green dye, ensuring stable fluorescent labeling. They are suitable for various applications, providing a long-lasting and well-visible signal.
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Why is it challenging to detect exosomes through immunofluorescence?
Exosomes are so small that it is difficult to get sufficient antibody binding for their detection via immunofluorescence. However, transmission electron microscopy has been performed on exosomes.
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What is the ideal exosome yield per cell?
Certainly, exosome secretion is indeed influenced by cell death. Therefore, it is highly advisable to utilize healthy and viable cells when studying exosomes. Furthermore, it is worth noting that apoptotic cells tend to release a significant amount of apoptotic vesicles.
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How can I visualize apoptotic bodies, microvesicles, and the exosome pellet obtained through centrifugation?
After centrifugation, it is not possible to distinguish between exosome pellet and apoptotic body pellet. To characterize individual extracellular vesicles (EVs), techniques such as Western blotting (WB), flow cytometry (FACS), electron microscopy, and atomic force microscopy can be used. For larger vesicles including apoptotic bodies, immunofluorescence imaging can be employed. To assess size distribution, nanoparticle tracking analysis and dynamic light scattering can be utilized.
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Which storage condition (4°C, -20°C, or -80°C) is optimal for preserving conditioned media prior to exosome isolation?
While we advise processing fresh samples whenever possible, cell media can be stored at 4°C for up to one week without significant changes observed in exosomes or extracellular vesicles (EVs). For long-term storage, it is recommended to store at -80°C.
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What is the anticipated concentration of RNA in serum exosomes?
Exosomes typically contain minimal amounts of nucleic acids. The concentration of RNA in serum samples is so low that it is not detectable on agarose gel or even by nanodrop spectrophotometry. Creative Biostructure provides sensitive, reproducible methods for exosome analysis, our exosome analysis services make it easy to profile exosome lipids, proteins, and RNAs.
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Can I use heparin or an EDTA tube to collect blood samples to isolate exosomes from plasma?
No. Heparin will significantly impair the downstream RNA assays. EDTA may interfere with the downstream PCR assay. Immediately centrifuge the sample to collect the plasma for exosome isolation.
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Can I block the release of exosomes from cancer cells?
Dimethyl amiloride (DMA), GW4869, and a Glucosyl ceramide synthase inhibitor (di threo-1-1phenyl-2-decanoylamino-3-morpholino-1-propanol) have demonstrated their ability to hinder exosome secretion in various types of cells. Alternatively, suppressing the expression of Rab27 or Rab35 through shRNA knockdown represents an excellent approach for inhibiting exosome release.
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What causes the elevated levels of exosomal miRNA in plasma compared to miRNA levels in serum exosomes?
During the preparation of serum samples, when removing the clot from whole blood, it is likely that exosomes and their associated miRNA are being lost. This could explain the disparity in exosomal miRNA levels between serum and plasma, as plasma samples have a lower chance of vesicle and exosomal miRNA loss.
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Does cell death affect exosomes?
The yield of exosomes can vary depending on several factors, including the cell type, culture conditions, isolation methods, and experimental protocols. Exosome yield is typically reported as the number of exosomes per cell or the concentration in a given volume of culture media. To obtain a more accurate estimate of the exosome yield per cell for a particular experimental system, it is recommended to consult relevant scientific literature or perform pilot experiments using the specific cell type, culture conditions, and isolation methods that you intend to use.
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How do you prepare exosome samples for western blot analysis?
For optimal detection of exosome proteins in western blot analysis, we suggest using non-reducing samples. This means that your samples should not contain reducing agents such as DTT or beta-mercaptoethanol during the boiling step prior to loading onto the PAGE gel. This is because the detection antibodies used for targeting tetraspanins in western blot analysis perform best under non-reducing conditions.
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Is there a distinct marker that can differentiate between apoptotic bodies and exosomes derived from the same cells?
Typical markers of apoptotic bodies such as C3b, Annexin V, and thrombospondin can be utilized to distinguish them from exosomes.
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What are the negative controls used for exosome isolation?
Negative controls are used in exosome isolation experiments to help identify and account for non-exosomal components that may co-purify with exosomes and affect experimental results.The following proteins must not be present in the exosome preparation: Grp94HSP90B1, mitochondria (cytochrome CCYC1), calnexin, Endoplasmic reticulum, Argonaute/RISC complex, Golgi (GM130), and nucleus (histones).
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Could you explain what Plasma Exosome Standards refer to?
Plasma exosome standards are purified lyophilized exosomes isolated from a pool of healthy donors by differential ultracentrifugation and microfiltration.
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Does the freezing affect the purified exosomes?
Fresh exosome samples are ideal but frozen samples at -80°C can be used if they were frozen immediately after isolation, have not been thawed multiple times, and have not been frozen for too long. Exosomal membrane is different from cell membrane and contains phospholipids, sphingolipid, ceramides and tetraspanin proteins (CD63, CD81, CD9) that help maintain its stability. However, freeze-thaw cycles can affect the size and morphology of exosomes, which may impact size determination in studies using techniques such as DLS, NTA, bioassays, and EM.
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For studying exosomes in serum samples, is the use of DMSO necessary?
DMSO is used to protect cell membranes when freezing, but it's not recommended to use it when freezing serum samples at -80°C because the small size of exosomes and liquid content makes it unnecessary.
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How to determine the protein concentration in exosomes?
After lysing the exosomes, you can use the Bradford or BCA methods to detect the protein concentration in them, which will provide the protein concentration amount in μg/μl.
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Is there any specific exosome biomarker?
There is currently no widely agreed upon universal exosome marker. It is strongly advised to use multiple exosomal markers such as CD9, CD81, and CD63 to confirm the exosomes.
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What is the difference between exosomes and microvesicles?
The diameter of exosomes is smaller than that of microvesicles, the diameter of exosomes is between 30-120 nm, while the diameter of microvesicles is between 100-1000 nm. Furthermore, they have different markers and play different roles in genetic exchange and cellular communication.
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Can I freeze purified exosomes after isolation?
Fresh samples are always better than frozen samples. However, samples frozen at -80 °C can also be used, provided they are frozen immediately after isolation, without multiple freeze-thaw cycles and for relatively short periods of time. Freeze-thaw cycles may affect exosome morphology and size determination studies.
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How to isolate and purify exosomes from tissue samples?
Since exosomes are secreted, we recommend performing short-term tissue explant cultures to isolate exosomes from tissues. You can collect cell supernatants and isolate exosomes using a protocol similar to the protocol for exosome isolation from cell culture media.
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Why specific isolation of exosomes is important?
Specific isolation of exosomes is important because exosomes play a crucial role in intercellular communication and can carry biological information, such as proteins and RNA, from the producing cells to the recipient cells. Therefore, the specific isolation of exosomes allows for the study of their contents, which can provide insights into cellular processes and potentially lead to the discovery of new therapeutic targets for various diseases. However, it is also important to note that exosomes can be mixed with other extracellular vesicles, such as apoptotic bodies and microvesicles, making specific isolation necessary to ensure accurate characterization and interpretation of results.
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What are the methods of exosome quantification?
There are several methods for quantifying exosomes, including Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), Enzyme-Linked Immunosorbent Assay (ELISA), Western blotting, Flow cytometry, RNA quantification using real-time PCR, Mass spectrometry (MS). Each method has its strengths and limitations, and the choice of method depends on the specific research question and the available resources.
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What biomarkers do you use to identify the isolated exosomes?
We use a combination of exosomal markers such as Alix 1, CD9, CD63, CD81, flotillin and TSG101 to characterize the isolated exosomes.
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Is there any specific biomarker for exosomes isolated from adipocytes?
It has been shown that almost all adipocyte-derived exosomes express FABP4.
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How should I store exosome products?
Our exosome products are usually in the form of lyophilized powder or frozen liquid. Lyophilized powder store at 4 °C. Frozen liquid store at -20°C to -80°C. Please avoid freeze/thaw cycles and keep them under optimal storage conditions as described in the instructions.
Liposome Products
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Can you provide information on the size and composition of your liposomes?
Yes, we offer detailed specifications for each product, enabling researchers to select liposomes that best match their experimental requirements.
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Do you offer customization options for liposome formulations?
Yes, we offer customization services to meet the unique demands of research projects. Our team of experts can work with customers to develop custom liposome formulations tailored to their specific needs, ensuring optimal results for their experiments.
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What analytical and characterization data do you provide with your liposome products?
We provide comprehensive analytical and characterization data for all our liposome products, such as size distribution, zeta potential, and encapsulation efficiency.
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How do you stay abreast of advancements in liposome research?
Our team closely monitors scientific developments and collaborates with experts in the field. This proactive approach allows us to update our product offerings regularly, incorporating the latest innovations and ensuring that researchers have access to cutting-edge liposome technologies for their experiments.
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Do you offer customization options for liposome products?
Yes, we offer customization options for our liposome products. You can discuss your specific requirements with our customer service team to explore options for customizing liposome sizes, surface modifications, or encapsulation of particular molecules.
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How do you ensure that the drug is effectively retained within the liposomes?
The encapsulation efficiency of our drug-loaded liposomes is crucial. We carefully optimize our formulation and manufacturing processes to achieve high encapsulation efficiency.
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What methods do you use to characterize the size and distribution of your liposomes?
We employ advanced techniques such as dynamic light scattering (DLS) and electron microscopy to accurately characterize the size and distribution of our liposomes.
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Do you offer any customization options for drug loading capacity or specific lipid compositions in your drug loaded liposomes?
Yes, our team is available to discuss your specific requirements and work with you to tailor liposomes to your research goals.
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How do Liposomes improve the effectiveness of skincare formulations?
Liposomes in skincare formulations can enhance the penetration of active ingredients through the skin barrier. This improved delivery can lead to better hydration, reduced signs of aging, and overall improved skin health.
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Can liposome products be customized for specific applications or formulations?
Yes, our liposome products can often be customized to meet specific formulation requirements. Our team can work with you to tailor liposomes for your unique needs, such as adjusting vesicle size, encapsulating specific molecules, or optimizing release profiles.
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How can I place an order for liposomes, and what is the minimum order quantity?
To place an order for liposome products, you can contact our sales team through our website or directly by phone or email. MOQ may vary depending on the product and your specific requirements. Our sales team will provide you with all the necessary information to start the ordering process.
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Are there any specific advantages of liposomes for dietary supplements compared to traditional forms of supplements?
Liposomes for dietary supplements offer advantages such as improved absorption, reduced gastrointestinal discomfort, and enhanced bioavailability of nutrients. These benefits can result in more effective and efficient nutrient delivery compared to traditional supplement forms.
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How do you ensure batch-to-batch consistency in liposome production?
Our scientists have detailed knowledge of the physical and chemical properties, normal operating ranges, and parameters of liposome products, which facilitate consistent production between batches. Additionally, our custom liposome service supports characterization during the manufacturing process, so any batch-to-batch inconsistencies can be detected.
Membrane Proteins & Nanodiscs
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What are the key advantages of pre-assembled nanodiscs?
Pre-assembled nanodisc products offer a streamlined solution for membrane protein studies. They provide a ready-to-use platform, reducing preparation time and ensuring consistent results.
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Can you elaborate on the compatibility of pre-assembled nanodiscs with common analytical techniques?
Pre-assembled nanodiscs are compatible with a range of analytical techniques, including NMR spectroscopy, circular dichroism, and various chromatographic methods. Their well-defined structure facilitates accurate analysis of membrane protein properties, allowing researchers to obtain detailed insights into structure, dynamics, and interactions.
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What quality control measures are in place for pre-assembled nanodiscs?
Quality control measures for pre-assembled nanodiscs typically include rigorous testing of stability, homogeneity, and functionality. We can provide detailed QC data with each product batch.
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What considerations should be kept in mind regarding the selection of MSPs?
Researchers should consider factors such as the size, charge, and topology of the target membrane protein when selecting membrane scaffold proteins. Tailoring the choice of MSP to match the characteristics of the protein of interest ensures optimal stabilization and functionality. Additionally, consulting literature and available protocols for recommended MSP-protein combinations can enhance experimental success.
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Are there specific applications where assembled nanodiscs excel?
Assembled Nanodiscs excel in membrane protein studies, drug delivery systems, and structural biology due to their stability, customizable lipid composition, and compatibility with diverse biomolecules, offering researchers a reliable platform for robust and insightful results.
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What is the pricing and are there bulk purchase discounts for Nanodiscs products?
Our pricing for Assembled Nanodiscs is transparent and competitive. For specific product pricing and potential bulk purchase or long-term collaboration discounts, contact our sales team directly for personalized solutions.
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How can MSP benefit my research?
Membrane Scaffold Proteins (MSPs) play a vital role in stabilizing and organizing membrane proteins, facilitating structural and functional studies. By creating a stable environment, our products enhance research precision, leading to more reproducible and higher-quality findings.
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What support accompany your membrane scaffold proteins?
In addition to our high-quality membrane scaffold proteins, we provide extensive technical support, documentation, and application resources. We aim to assist researchers throughout, from product selection to troubleshooting, ensuring a seamless integration of our products into your experiments.
Virus-like Particle Products
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What expression systems do you use for your VLP products?
Our virus-like particles are produced from different expression systems such as E. coli, yeast, insect cells, and HEK293 cells.
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What are the key factors that determine the success of membrane protein expression?
It depends on several factors, including the choice of expression system, the target membrane protein's characteristics, and optimization of culture conditions. Our team employs state-of-the-art techniques to carefully select the most suitable expression system based on your protein's properties, ensuring high yields and functionality.
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Can you provide insights into the timeline involved in membrane protein expression projects?
The timeline for membrane protein expression projects varies based on the complexity of the target protein. Our experienced team establishes clear milestones, considering cloning, expression optimization, and purification steps. We prioritize efficient timelines without compromising the quality of expression, ensuring timely delivery of results.
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What strategies do you employ for overcoming challenges during membrane protein expression?
Membrane protein expression can face challenges such as low expression levels or protein misfolding. Our team is equipped with a comprehensive troubleshooting approach, utilizing advanced techniques like codon optimization, co-expression with chaperones, and optimization of lipid compositions. This ensures successful expression and maintains protein integrity.
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Can you accommodate specific modifications or custom requirements in membrane protein production?
Certainly. Our custom membrane protein service is tailored to meet your specific needs, and we will work closely with you to ensure the final product aligns perfectly with your research goals.
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What's the confidentiality for our custom membrane protein project's proprietary information?
We prioritize confidentiality, handling your proprietary information with care and strict protection. Our team adheres to rigorous confidentiality agreements. Trust us to safeguard your intellectual property throughout the custom membrane protein production process.
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How can your custom membrane protein production cater to my specific research needs?
Our custom membrane protein production service is tailored to meet your research demands. We collaborate closely, understanding objectives to design a customized production plan. This ensures precise alignment with your goals, optimizing research success.
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What steps should be taken to address DNA contamination in the purification process of full-length protein?
Size exclusion chromatography (SEC) is typically effective for isolating proteins from DNA, and if needed, ion exchange chromatography can be explored as an additional step.
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The gene product is cytotoxic, how to express the protein?
If you have determined this, you can try the cell-free expression system.
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How to interpret three-dimensional protein?
Interpreting a 3D protein structure involves visualizing the structure using software like PyMOL, identifying its overall fold, secondary structures, and active sites. Analyzing ligand binding, protein-protein interactions, and solvent accessibility provides insights into function and stability. Consider flexibility and dynamic regions, and use comparative analysis or homology modeling if needed. Collaboration with our experts is valuable for a comprehensive interpretation.
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Can a protein in gel filtration appear to be smaller than its actual size?
Yes, if a protein has an unusual shape, it might behave differently than expected in gel filtration chromatography. For example, a protein that is elongated or has an extended structure might enter the pores of the gel to a greater extent than a more compact protein of the same molecular weight. This can lead to the elongated protein eluting later and appearing larger on the chromatogram than the more compact protein.
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What is the minimum concentration of urea required for a protein to denature?
The concentration of urea required for denaturation will depend on the protein's stability. Some proteins are more stable and require higher concentrations of urea to denature, while others are less stable and can denature at lower urea concentrations. In general, denaturation is often achieved in the range of 4 to 8 M urea for many proteins.
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What is the most effective method for concentrating very low molecular weight proteins or peptides?
If you want to maintain the protein's activity to the greatest extent, it's recommended to use an ultrafiltration membrane system with a molecular weight cutoff (MWCO) of 1 kD or 3 kD. Additionally, using a lower centrifugal speed will help ensure that the target protein does not pass through the membrane or aggregate. Alternatively, you can use a dialysis bag with the appropriate MWCO range, but please be aware that proteins may aggregate and precipitate during dialysis.
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Can centrifugation influence weak protein-protein interactions?
High-speed centrifugation for extended periods may have a greater impact on weak interactions compared to lower speeds and shorter durations.
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Could you please recommend techniques for identifying the specific region of interaction in one of the protein-protein interacting partners?
To pinpoint the precise interaction site, we recommend employing structural biology techniques such as Nuclear Magnetic Resonance (NMR), X-ray Crystallography and Cryo-Electron Microscopy (Cryo-EM). Each of these methods comes with its own set of strengths and weaknesses, and our team can assist clients in selecting the most suitable approach based on the properties of the target protein.
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How to express a protein at the cell surface?
To express a protein at the cell surface, select the target protein, clone it into an expression vector with a signal sequence, transfect or transduce it into the chosen cell line, ensure proper protein trafficking, confirm expression at the cell surface, and perform functional assays as needed.
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How to study protein-peptide interactions?
You can try SWISS-MODEL or PyMol for preliminary predictions. However, if you require precise information about the interaction sites, the structural biology technologies, such as X-ray crystallography, NMR, and cryo-electron microscopy technology, must be used to solve this problem.
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Can I add fresh DTT and PMSF to previous buffers?
DTT and PMSF can be added to the buffer without contamination. However, the precise concentration of DTT and PMSF within the buffer remains uncertain. This uncertainty could potentially lead to irreproducible results in subsequent experiments, particularly when purifying proteins for crystallization, a process that demands meticulous attention to detail. It is suggested to initially prepare the buffer without DTT and PMSF. Subsequently, prior to conducting the experiment, the buffer can be adjusted and DTT and PMSF can be added proportionally.
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How much time is sufficient to remove most of the salt from a protein-containing buffer using dialysis?
It varies depending on several factors, including the volume of the buffer, the initial salt concentration, the size of the protein, and the type of dialysis membrane being used. In general, dialysis can take several hours to overnight for small volumes of protein-containing buffer (e.g., 1-10 mL) to remove the majority of salt. There is no fixed time that universally applies, and it's recommended to perform pilot experiments to determine the optimal dialysis time for your specific buffer, protein, and desired salt removal level.
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How can I get a cDNA library for membrane proteins?
To fulfill your experimental needs, you can explore yeast random display technology and phage display technology. These methods offer valuable insights. If you need technical support, Creative Biostructure can provide related services.
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Is it feasible to express the spike protein of the PED virus using the E.coli system, specifically PET 24a or PET 32 expression vectors?
The E. coli expression system is not optimal for preserving the activity and native conformation of eukaryotic-derived proteins. If feasible, it is recommended to utilize eukaryotic expression systems, despite the slightly more complex construction of the expression system. This approach would greatly benefit subsequent experiments.If you don't have enough conditions, you can consult Creative Biostructure, we provide professional protein expression services.
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Does the addition of restriction sites to my construct impact peptide folding?
It is advisable to select a restriction site that encodes for Glycine (Gly) or Serine (Ser), while avoiding large amino acids that might introduce steric hindrance. Additionally, it is recommended to predict in advance whether the introduction of these amino acids alters the secondary structure of your target protein.
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Why am I getting slimy bacterial pellet after protein expression?
It may be that your cells are not growing well, probably because of phage contamination or the toxicity of the expressed target protein.
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Molecular docking tools and ligand-protein interactions scoring?
There are some free academic molecular docking software: AutoDoc (khttp://autodock.scripps.edu/), rDock (http://rdock.sourceforge.net/), and UCSF DOCK (http://dock.compbio.ucsf.edu/).
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What are the factors affecting recombinant protein activity and stability?
1. Expression system: The choice of expression system can affect the quality, quantity, and post-translational modifications of the protein. Different expression systems have different advantages and disadvantages, and the choice of system depends on the specific protein being produced.
2. Post-translational modifications: Some recombinant proteins require specific post-translational modifications, such as glycosylation or phosphorylation, for proper folding, stability, and activity. The absence or incorrect modification of these post-translational modifications can affect protein function.
3. pH and temperature: Proteins are sensitive to changes in pH and temperature, and exposure to extremes can denature the protein or lead to aggregation. Optimal conditions for protein activity and stability vary depending on the protein and can be determined experimentally.
4. Oxidative stress: Oxidative stress can lead to the formation of disulfide bonds, which are critical for protein stability, but can also lead to the formation of protein aggregates or misfolding.
5. Proteolysis: Proteins can be subject to degradation by proteases, which can affect protein activity and stability. Protection of the protein from proteolysis can be achieved by incorporating protease inhibitors.
6. Aggregation: Recombinant proteins can aggregate, which can reduce protein activity and stability. Aggregation can be caused by a variety of factors, including pH, temperature, and protein concentration. Measures such as optimization of protein production, use of chaperones, or chemical modification of the protein can prevent or reduce protein aggregation. -
Can you provide guidance on selecting SDS gels (both stacking and separating gel) that are appropriate for proteins of varying sizes?
The percentage of the separating gel will depend on the size of the protein of interest. Lower percentages of the gel (e.g., 8%) are suitable for larger proteins (>200 kDa), while higher percentages (e.g., 15%) are suitable for smaller proteins (<50 kDa). For proteins in the intermediate size range (50-200 kDa), a gel percentage between 10% and 12% is commonly used. The percentage of the stacking gel is generally lower than that of the separating gel and is typically between 4% and 5%.
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Does my protein sample degrade after being left overnight at room temperature?
It is possible that your protein sample may degrade if left overnight at room temperature, depending on the specific properties of the protein and the conditions under which it was stored. To determine whether your protein sample has degraded, you may need to perform various assays or tests, such as gel electrophoresis, Western blotting, or enzyme activity assays.
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Why can't my size exclusion chromatography column separate my 15kD and 31kD proteins?
1. Column equilibration: If the column is not equilibrated properly, the proteins may not bind or elute from the column as expected, leading to poor separation.
2. Column size/pore size: If the column is not optimized for the size range of your proteins, you may not see good separation.
3. Protein-protein interactions: It is also possible that your proteins are interacting with each other, forming dimers or other higher-order complexes. If this is the case, you may need to use a different chromatography method that is better suited for your protein system, such as ion exchange.
4. Sample preparation and purity: If the sample contains impurities, such as other proteins or contaminants, this may affect the separation. Ensure that your protein sample is pure and well-prepared before loading it onto the column. -
How can point mutations be used to obtain higher protein expression than the original gene?
Point mutations can be used to increase protein expression levels by optimizing codon usage, modifying the promoter region, and stabilizing the protein structure. However, it's important to note that point mutations can also have negative effects on protein function, so careful experimentation and analysis are necessary to determine the optimal mutation strategy for a particular protein.
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Why am I losing all of my target protein during purification?
1. Protein degradation: Your target protein may be degraded during the purification process. This can happen due to the presence of proteases or other enzymes in the sample, or as a result of prolonged exposure to heat, pH extremes, or other harsh conditions during the purification process. It is recommended that you use 0.5 mM TCEP during the purification process.
2. Poor solubility: Your target protein may be insoluble or poorly soluble in the buffer conditions you are using, leading to precipitation or aggregation of the protein. This can result in the loss of the protein during purification.
3. Non-specific binding: Your target protein may be sticking to surfaces of the purification column, tubes or other equipment, resulting in non-specific binding and loss of the protein during the purification process.
4. Inefficient purification strategy: The purification strategy you are using may not be optimized for your target protein. Different proteins require different purification strategies and conditions to maximize recovery and purity.
5. Incorrect sample preparation: Improper sample preparation such as incorrect sample dilution, homogenization or centrifugation steps can result in loss of the protein.
6. Experimental error: There can be experimental errors such as inaccurate measurements, incorrect buffer preparation, or pipetting errors, leading to loss of the protein. -
What are the requirements for crystalline grade recombinant protein?
For a recombinant protein to be considered 'crystalline grade', it typically needs to meet the following requirements:
1. Purity: The protein should be highly pure, with minimal contamination from other proteins, host cell proteins, or other impurities.
2. Homogeneity: The protein should be highly homogeneous, with minimal batch-to-batch variation in terms of its physical and chemical properties.
3. Solubility: The protein should be soluble in a physiologically relevant buffer, and should not aggregate or precipitate under the conditions used for crystallization.
4. Stability: The protein should be stable over time, retaining its physical and chemical properties even when stored for long periods of time.
5. Concentration: The protein should be at a high enough concentration for crystallization.
6. Expression level: The protein should be expressed at a high enough level to allow for purification and crystallization in a reasonable time frame.
Meeting these requirements helps ensure that the recombinant protein is suitable for structural studies, such as X-ray crystallography or cryo-electron microscopy, that require high-quality samples. Creative Biostructure's one-stop service can help you realize Gene-to-Structure, which is more worry-free and efficient. -
What are the fusion tags that promote protein solubilization?
For example, His-tag, GST-tag, MBP-tag, SUMO-tag, and TAP-tag. These tags contain amino acid sequences that allow the target protein to be efficiently purified or stabilized, while also promoting its solubility in aqueous environments.
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Can a prokaryotic protein be expressed extracellularly using E.coli (DH5-α)?
The DH5-α series of plasmids are used to clone the target gene and are not suitable for use as expression vectors. Alternatively, if your target protein needs to be expressed into the periplasm or extracellular, you can choose to use the pET-22b vectors.
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Is there any method to identify whether a protein has a monomeric or oligomeric structure?
This can first be demonstrated initially by combining gel filtration and SDS-PAGE results, and structural biology approaches can also be considered if high-purity proteins can be extracted.
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Why are target proteins not expressed?
1. Careful checking the cloned sequence, not only to check the gene sequence encoding the target protein but also to pay attention to whether the position of the target gene inserted into the expression vector is correct.
2. Temperature, time, and IPTG concentration will affect the expression of the protein. It is suggested that you can set the gradient conditions according to these factors to conduct experiments and compare the expression under different conditions.
3. Prokaryotic expression system may lead to incorrect folding or instability of eukaryotic proteins. Methods such as codon optimization and chaperones can be tried. Furthermore, it is recommended to change the expression vector or even the expression system, such as insect cells or mammalian cells. -
I encountered aggregation when using E.coli to induce the expression of proteins. What should I do?
It is necessary to consider whether the IPTG concentration is appropriate and whether the induced temperature is relatively high, etc. In addition, protein aggregation is mainly due to misfolding, and you might consider using chaperones to help proteins fold.
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Methods to quantify the protein-protein interaction?
Quantitative methods in vitro: Surface Plasmon Resonance (SPR), and Isothermal Titration Calorimetry (ITC). Structural methods: NMR Spectrometry, X-ray Crystallography.
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How do you handle data interpretation and analysis for X-ray crystallography results?
Our team includes experts in structural biology who are proficient in interpreting X-ray crystallography results. We provide detailed structural analysis reports, highlighting key structural features and interactions. Clients can expect comprehensive insights into the three-dimensional arrangement of atoms, aiding in a deeper understanding of the protein's function.
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How do you ensure the quality of X-ray crystallography data in your services?
Our X-ray crystallography services prioritize quality and reproducibility. We adhere to rigorous crystallographic standards, implement automated data collection techniques, and perform extensive validation procedures. This ensures that the crystallographic data obtained is of high quality and can be reliably reproduced.
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How long does it take to receive X-ray crystallography results?
The turnaround time for X-ray crystallography results can vary based on the complexity of the project and the specific requirements. Typically, we strive to deliver results promptly without compromising the quality of the analysis.
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Why crystal are not forming?
To enhance your chances of protein crystallization success, explore a broader range of screening conditions. Understand your target protein's properties, considering factors like pH, buffer concentration, and temperature, and ensuring purified protein is free of aggregation. If the current expression and purification conditions prove insufficient, consider constructing alternative forms or utilizing different expression systems. If these efforts are unsuccessful, explore structural biology methods that do not rely on crystallization.
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How to increase solubility of protein for crystallization?
To increase the solubility of a protein for crystallization, optimize protein expression and purification, adjust pH and buffer conditions, experiment with salts or cosolvents, consider the use of molecular chaperones, utilize fusion tags, try different crystallization conditions, concentrate the protein, and store it in small aliquots. These strategies are protein-specific and may require multiple trials, and protein engineering or co-crystallization can also be considered to enhance stability.
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How to co-crystallize a protein with its ligand?
Co-crystallization of a protein with its ligand is a crucial step in structural biology for determining the three-dimensional structure of the protein-ligand complex. The process involves expressing and purifying the protein, preparing the ligand, conducting initial crystallization screenings, optimizing crystallization conditions, setting up co-crystallization experiments, growing crystals, harvesting them, collecting X-ray diffraction data, solving the structure, and refining it. While this process can be challenging, our experienced crystallographers and specialized equipment and facilities greatly boost success rates.
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If the ratio of A280 to A260 is about 1.2, does this mean that my protein has DNA contamination? And does this affect crystal growth?
A ratio of A280/A260 around 1.2 suggests that there might be some nucleic acid contamination in your protein sample, particularly DNA contamination. However, it's important to note that the specific interpretation of the ratio can vary depending on factors like the buffer used, the presence of other contaminants, and the type of protein you are working with. Regarding the impact on crystal growth in protein crystallization, nucleic acid contamination can indeed affect the crystallization process. Nucleic acids have different physical and chemical properties compared to proteins, and their presence can interfere with the delicate process of crystallization. This interference can lead to difficulties in obtaining high-quality protein crystals suitable for X-ray crystallography. To mitigate this issue, it's generally recommended to further purify your protein sample to reduce nucleic acid contamination. Techniques such as size exclusion chromatography, ion exchange chromatography, or DNA/RNAse treatment can be employed to achieve higher protein purity, which in turn may improve the success of crystallization experiments.
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What is the optimal concentration for preparing crystallization expansion plates?
A commonly used starting point for crystallization experiments is to prepare a concentrated stock solution of the substance you want to crystallize. For protein crystallization, the typical concentration range for setting up initial screening conditions is around 5-30 mg/mL. This concentration range allows for a good chance of nucleation and crystal growth while providing enough material for subsequent optimization steps. It's important to note that these concentration ranges are general guidelines, and optimization of crystallization conditions is often necessary to obtain high-quality crystals suitable for structural determination or other applications. Factors such as pH, temperature, precipitant concentration, and the presence of additives may also need to be considered and optimized during the experimental setup.
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What is the ideal concentration for setting up crystallization expansion plates?
The ideal concentration for setting up crystallization expansion plates can vary depending on the specific system and the type of crystals you are trying to grow. Therefore, it is recommended to consult the literature specific to your molecule or seek guidance from experienced crystallographers or structural biologists who have expertise in the particular system you are working with.
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Does presence of sarkosyl affect protein crystallization?
Yes, the presence of sarkosyl can affect protein crystallization. Sarkosyl is a detergent commonly used in protein purification to solubilize membrane proteins or remove lipids. While sarkosyl can be useful in maintaining the solubility of certain proteins, it can also have an impact on the crystallization process. The concentration of detergent molecules in the experimental operation will affect the stability of membrane proteins, and the hydrophobic region of the membrane protein wrapped by the detergent hinders the interaction between the membrane protein molecules, in turn, affects the lattice order to pile up. Therefore, the determination of the type and concentration of detergent is critical in the study of membrane protein crystallization.
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How can I improve crystal diffraction?
1. Increase the quality of the crystal: The quality of the crystal is crucial for obtaining good diffraction patterns. The crystal should be as large, pure and well-ordered as possible. If the crystal is not of good quality, it may diffract poorly or not at all.
2. Use multiple data collection strategies: Collecting data from different crystal orientations and using different data collection strategies (such as using synchrotron radiation) can improve the quality of the diffraction pattern.
3. Improve data processing and refinement: The data collected from the diffraction experiment must be processed and refined to obtain an accurate atomic model. Improving the software used for processing and refinement can improve the accuracy and reliability of the final atomic model.
4. Consider alternative methods: In some cases, alternative methods such as electron crystallography may be more appropriate for a specific crystal or for obtaining specific structural information. -
Does the presence of sarkosyl affect protein crystallization?
Sarkosyl (N-lauroylsarcosine) is a detergent commonly used to solubilize and extract membrane proteins. However, its effect on protein crystallization can be variable and depends on the specific protein being studied and the conditions used for crystallization. Generally speaking, changes in the concentration of detergents used in experimental procedures can affect the stability of membrane proteins. The hydrophobic region of the membrane protein wrapped by the detergent hinders the interaction between the membrane protein molecules, in turn, affects the lattice order to pile up.
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Why does my dimer target protein appear as a monomer during crystal growth?
It is possible that the dimeric form of your protein may be unstable or only exist transiently in solution, and that the monomeric form is the dominant form under the conditions used for crystal growth. In this case, obtaining dimeric crystals may be challenging, and you may need to explore other approaches, such as co-crystallization with binding partners or the use of protein engineering to stabilize the dimeric form.
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Why the protein crystals do not come out with the original condition?
1. Improper handling or poor sealing can easily cause reservoir contamination.
2. Use the same buffer, salt, and precipitant as in the initial screening kit, including the conductivity of the ddH2O used to prepare the buffer.
3. Inoculate the previously obtained crystals into droplets used to optimize the crystal to promote nucleation. -
What if my target protein cannot crystallize?
1. Try as many screening conditions as possible.
2. Understand the nature of your target protein and analyze which buffer system is more suitable for your protein, Tris/MES/Hepes...
3. It may be that the current expression and purification conditions cannot guarantee that your protein used for crystallization is in a stable state, so it is recommended that you try more constructs and expression systems.
4. If none of the above attempts can solve your problem, there are other techniques that can be used to study its structure, such as NMR spectroscopy and cryo-EM. -
I want to elucidate a protein structure, should I choose X-ray crystallography or NMR?
This article compares the advantages and disadvantages of these two methods, and maybe can help you choose the experimental method for determining the structure: Comparison of Crystallography, NMR and EM.
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What is the difference between protein structure determined by X-ray crystallography and NMR?
Both X-ray crystallography and NMR are important techniques for determining protein structure, but X-ray crystallography gives higher resolution structures while NMR provides information on solution structures and dynamics.
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Difference between protein structure determined by cryo-EM and X-ray crystallography?
Cryo-EM is best suited for studying flexible or heterogeneous protein complexes, while X-ray crystallography is best suited for studying highly ordered, crystalline proteins.
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Why is it best to use fresh protein solutions for crystallization?
To guratee the activity of the protein during crystallization, please try to crystallize the protein immediately after purification and ensure that the process of protein purification is efficient to reduce the risk of protein degradation or aggregation.
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If I cannot crystallize the protein immediately after purification, how can I ensure the freshness of the protein?
It is recommended that you freeze the purified protein in liquid nitrogen and store it below -70°C to ensure maximum protein behavior.
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I am currently using the hanging drop crystallization method, any suggestions to facilitate nucleation?
In general, the nucleation rate of the hanging drop method is slower than that of the sitting drop method, but the crystals formed are larger and more regular, which is more conducive to diffraction. If you want to speed up nucleation, try the 18℃ condition, or inoculate the crystals from the sitting drops into the droplets of the hanging drops to promote nucleation.
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How to distinguish protein crystals from salt crystals?
In the case of the known color and shape of target protein crystals and sodium chloride crystals, direct discrimination can be observed with a polarizing microscope. And the symmetry of the corresponding crystal can be judged.
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What are 'crystallographic grade' proteins?
The protein for crystallization should be homogeneous and of high purity (>90%). The purity of the protein should be checked by HPLC or MS prior to crystallization. The minimum amount is 10ug/ul x 300ul.
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What is the typical turnaround time for MicroED projects?
Turnaround time varies based on project complexity. We prioritize efficient data collection and processing while maintaining high-quality standards. Our streamlined workflows and optimized protocols enable us to deliver results in a timely manner without compromising the accuracy and reliability of MicroED data.
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How experienced is your team in handling cryo-EM projects?
Our team comprises highly skilled scientists and technicians with extensive experience in cryo-EM. We have successfully undertaken a diverse range of projects, demonstrating proficiency in sample preparation, data collection, and image processing to meet the highest scientific standards.
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What types of samples are compatible with your cryo-EM service?
Our cryo-EM service accommodates a wide range of samples, such as proteins, complexes, viruses, and cellular structures. Our team will work closely with you to ensure that your specific sample requirements are met.
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What is the typical turnaround time for Cryo-EM data acquisition and analysis?
Turnaround time for Cryo-EM services can vary based on factors such as sample complexity, data quality requirements, and instrument availability. Typically, data acquisition can take several days to weeks, while data analysis may require additional time.
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Can Cryo-EM services assist in determining macromolecular interactions within my sample?
Yes, Cryo-EM is a powerful tool for studying macromolecular interactions. By visualizing structures in their native state, this technique provides insights into how molecules interact within a cellular context. Whether studying protein-protein interactions, protein-nucleic acid complexes, or large cellular assemblies, Cryo-EM can reveal details about the architecture and dynamics of these interactions, aiding in the comprehensive understanding of biological processes.
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What is the typical turnaround time for a cryo-EM service, and can it be expedited if needed?
The timeframe varies based on project complexity, typically spanning from weeks to a few months. If you need quicker results, we offer expedited services at an additional cost. Contact us to tailor a solution to your specific project.
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What is the level of confidentiality and data security you provide for cryo-EM projects?
All project-related data is handled with care and is accessible only to authorized personnel. We are also open to signing non-disclosure agreements (NDAs) if needed to further safeguard your project's confidentiality.
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How is sample preparation for Cryo-EM different from other electron microscopy techniques?
Sample preparation for Cryo-EM is unique in that it involves rapid freezing of specimens in vitreous ice to preserve their natural state. This is in contrast to traditional electron microscopy techniques that often require staining, sectioning, or heavy metal contrast. Cryo-EM samples must be kept at cryogenic temperatures throughout the imaging process to prevent radiation damage.
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Can Cryo-EM be used for studying live cells or in situ structures?
Yes, Cryo-EM can be adapted for studying live cells or in situ structures using techniques like cryo-electron tomography (cryo-ET). Cryo-ET allows researchers to visualize cellular structures within intact cells or tissue sections at nanometer-scale resolution. This is particularly useful for understanding the 3D organization of cellular components in their native environment.
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What are some recent technical advancements in Cryo-EM that have contributed to higher resolution and improved structural details?
Recent advancements include the development of direct electron detectors with higher sensitivity and faster readout speeds, enabling the capture of higher quality images. Additionally, improvements in computational algorithms for image processing and the use of advanced data collection techniques like single-particle analysis and cryo-electron tomography have led to enhanced resolution and improved structural insights.
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How has Cryo-EM contributed to our understanding of complex biological processes, and can you provide a notable example?
Cryo-EM has significantly advanced our understanding of various biological processes by revealing high-resolution structures of intricate molecular assemblies. An exemplary case is the elucidation of the ribosome structure during translation. Cryo-EM studies have provided detailed insights into the dynamic conformational changes of ribosomal components and their interactions with mRNA and tRNA, shedding light on the molecular mechanisms of protein synthesis.
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What are the major challenges in sample preparation for cryo-EM?
Sample preparation for cryo-EM involves several challenges, such as maintaining sample integrity and minimizing artifacts. Key issues include preventing sample contamination, achieving proper grid preparation, optimizing vitrification to preserve the native structure, and managing sample heterogeneity.
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How can cryo-EM data be validated for structural determination?
Cryo-EM data validation involves several steps to ensure the reliability of the structural information obtained. This includes assessing the quality of the reconstructed maps through metrics such as Fourier Shell Correlation (FSC) and resolution estimation. Model building and refinement against the cryo-EM map can further validate the structural interpretation. Additionally, cross-validation with other experimental techniques, such as X-ray crystallography or biochemical assays, can provide additional validation and confirmation of the cryo-EM-derived structure.
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How is Cryo-EM contributing to drug discovery and development?
Cryo-EM has emerged as a valuable tool in drug discovery and development. It allows for the visualization and characterization of drug targets, such as membrane proteins and protein complexes, in their native states. This enables structure-based drug design, where potential drug candidates can be designed to interact with specific regions of the target protein. Cryo-EM also aids in the understanding of drug-target interactions and the mechanism of action of drugs, facilitating the optimization of drug efficacy and minimizing off-target effects.
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Can Cryo-EM be used to study protein dynamics?
Yes, Cryo-EM can be used to study protein dynamics. Recent advancements in Cryo-EM techniques, such as time-resolved Cryo-EM, have enabled the visualization of dynamic processes at high resolution. By rapidly freezing samples at different time points during a biochemical reaction or by using specialized sample holders, it is possible to capture distinct states of a protein or macromolecular complex. Computational analysis and data processing techniques can then be employed to generate movies or series of structures that reveal the conformational changes and dynamics occurring within the sample.
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How does cryo-EM compare to other structural biology techniques?
Cryo-electron microscopy has several advantages over other structural biology techniques, such as X-ray crystallography and nuclear magnetic resonance spectroscopy. Cryo-EM can visualize molecules in their native, biologically relevant state, without the need for crystallization or chemical modification. Cryo-EM can also image larger and more complex molecules than X-ray crystallography, making it particularly useful for studying viruses and large macromolecular complexes.
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What are some of the limitations of cryo-electron microscopy?
These limitations include technical challenges in sample preparation, the requirement for large amounts of protein, and limitations in resolution. At Creative Biostructure, we have developed innovative approaches to overcome these limitations, such as advanced image processing techniques to improve resolution and sample optimization strategies to minimize protein usage.
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What is the resolution limit of cryo-EM?
The resolution limit of cryo-EM is determined by a combination of factors, including the quality of the electron microscope, the quality of the sample, and the data processing methods used. Currently, cryo-EM can achieve resolutions down to 1.2-1.5 Å, which is approaching the resolution of X-ray crystallography.
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How does sample preparation affect the quality of cryo-EM data?
Sample preparation is critical for obtaining high-quality cryo-EM data. The sample must be flash-frozen in a thin layer of vitreous ice to preserve its native structure, and any contaminants or impurities in the sample can interfere with data collection. Additionally, the sample must be aligned in the microscope in a consistent and precise manner to obtain high-quality images.
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What is the difference between single-particle cryo-EM and cryo-electron tomography (cryo-ET)?
Single-particle cryo-EM and cryo-ET are two different methods of data collection in cryo-EM. Single-particle cryo-EM involves imaging many identical copies of a single macromolecular complex that are randomly oriented in the vitreous ice. The images are then processed to generate a 3D reconstruction of the complex. In contrast, cryo-ET involves imaging a sample at different tilt angles to collect a series of 2D projections of the sample from different perspectives. These projections are then combined to generate a 3D tomographic reconstruction of the sample. Cryo-ET is useful for studying complex biological structures that cannot be purified in sufficient quantities for single-particle cryo-EM.
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What are some common image processing techniques used in cryo-EM?
Common techniques used in cryo-EM image processing include Fourier transform, cross-correlation, and maximum-likelihood estimation. These techniques are used to align and average the particle images to improve their SNR, generate 3D reconstructions, and refine the atomic models.
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I want to use cryo-EM to determine protein structure, what are the requirements for sample preparation?
1. Purification: The protein sample should be pure and free from contaminants to minimize the noise in the images and improve resolution.
2. Concentration: The sample should be concentrated to a high enough concentration.
3. Vitrification: The sample must be rapidly frozen to a temperature below its glass transition temperature, forming a solid but amorphous glass, in order to preserve the native state of the protein.
4. Support: The sample should be placed on a thin film or grid to allow electrons to pass through and be captured by a detector.
5. Stabilization: The sample may need to be stabilized with cryoprotectants or detergents to prevent aggregation or denaturation during the preparation and imaging process.
These requirements should be optimized for each protein of interest to maximize the chances of obtaining high-resolution structures. At Creative Biostructure, you only need to provide the protein solution upon request, or tell us your target protein sequence, to achieve the research goal from sequence to structure. -
Can cryo-EM be used for drug discovery?
Yes, it provides high-resolution images of protein structures, which can be used to understand the molecular basis of diseases and to identify potential targets for drug design. Cryo-EM can also be used to study the mechanism of action of drugs and to determine the structures of drug-protein complexes, providing valuable information for the optimization of existing drugs and the development of new ones. Currently, Creative Biostructure offers you cryo-EM solutions for drug discovery.
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How can cryo-electron tomography be used to study membrane transport?
Compared to ordinary live-cell fluorescence microscopy, cryo-ET with high resolution not only captures the fast and dynamic events involved in the field of membrane trafficking, but also provides insight into the complex network structures that comprise the membrane trafficking system. Furthermore, by combining the cryo-ET technique with subtomogram averaging, we can obtain structures of specific protein complexes that bond to the membrane in a near-native state.
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What is the workflow for lamella preparation in cryo-ET?
In cryo-ET, the sample is first rapidly frozen, then thinned to a suitable thickness, and finally, a series of images are taken with a TEM. To thin the sample, a FIB/SEM has become the gold standard. FIB is utilized to sputter away the material and form a lamella.
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Why is cryo-EM technology important?
Cryo-EM allows us to observe proteins and other important structures without many of the limitations of traditional methods. For example, the protein expression level is not high, or the protein molecular weight is too large to crystallize. Since 2015, due to the improvement of cryo-EM technology, more and more cryo-EM structures have been reported.
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Which parameters need to be optimized for cryo-EM sample preparation?
There are many parameters to be optimized, including sample concentration, blot time, temperature, grid specifications (copper grid or gold grid), and a series of optimization conditions.
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Can I characterize liposomes under cryo-EM?
The size and shape of liposomes can be characterized by cryo-EM as it can directly visualize individual particles including their inner architecture.
