Pulldown

Protein pulldown assay takes advantage of a bait protein for target protein enrichment based on the interaction with the bait protein and target protein. It is an in vitro affinity purification method that can be used to confirm the existing protein-protein interactions, or to identify novel protein-protein interactions. Creative Biostructure provides technical support for protein pulldown as well as other techniques in protein purification, and we also have the most advanced instruments to perform protein pulldown with high-efficiency.

Pulldown assay is very similar to immunoprecipitation, only in which an antibody as the affinity reagent replaces the bait protein. The bait protein are usually with GST-tag, His-tag, or biotin-tag to capture or pulldown the target protein.

General Steps in a GST-tagged Protein Pulldown Assay

1. Choose the target protein;
2. Plasmid construction of GST-tagged protein;
3. Target protein expression;
4. Target protein purification;
5. Cross-linking of target protein to beads;   >>more
   1. a. Add proper volume of GST fusion protein beads into a column. Wash the column (beads to be cross-linked) with PBS buffer twice;
   2. b. Use cross-linker to cross-link the beads and column, then incubate under continuous mixing (45 min, room temperature);
   3. c. Drain the column;
   4. d. Add proper volume of quenching buffer into the column under gentle mixing (10 min, room temperature) to block amine-reactive sites;
   5. e. Wash the beads thoroughly at 4 °C.
6. Preparation of cell lysate or tissue;   >>more
   Make sure that lysate is free from contamination in case of resulting in large amounts of nonspecific protein. If nonspecific proteins present, you must get rid of them by high speed centrifugation.
7. Pulldown;   >>more
   1. a. Pre-clear lysate with GSH beads (This is extremely important if the tissue lysate contains large amounts of endogenous GSTs);
   2. b. Bind cell/tissue lysate to the GST fusion protein beads (Add appropriate volume of pre-cleared lysate into the column according to the volume of GST fused protein beads. 4 °C Incubation for 1 h with rotation);
   3. c. Recover the column and wash beads.
8. Elute proteins for SDS-PAGE analysis;
9. Mass Spectrometry Analysis;
10. Confirmation of the pulldown results with methods such as yeast two-hybird, surface plasmon resonance, co-immunoprecipitation, and fluorescence resonance energy transfer, etc.

Troubleshooting:

1. Interacting protein was not isolated.
If it dues to weak or transient interactions, you may decrease the ion concentration of the buffer or wash time;
If it dues to low-level expression of prey protein, please adjust your culture condition and increase the amount of the target protein.

2. Get large amount of nonspecific proteins after pulldown.
It may caused by the adhesion of hydrophobic proteins to the beads, you can add detergent such as Triton X-100 in the wash buffer to thoroughly wash the beads.

Besides pulldown, Creative Biostructure is also able to help your protein purification project with other technical resources. Please see the table below for more information and feel free to Contact Us.