GST Pull-down Kit
Experimental principle
GST Pull-Down experiment is based on GST (glutathione-S-transferase), which can bind to glutathione (GSH). GSH is fixed on magnetic beads to form GSH-magnetic beads. The known protein X is fused with GST and expressed. The obtained GST-X protein can bind to GSH-magnetic beads. If there is protein Y that interacts with protein X in the system, a "magnetic bead-GSH-GST-X-Y" complex will be formed, and the protein that interacts with protein X can be separated and detected.
Experimental Procedure
Reagent Components
|
Components |
Size (6T) |
Storage Temperature |
|
Lysis buffer |
9 mL |
4 ℃ |
|
Glutathione MagBeads |
200 μL |
4 ℃ |
|
Incubation buffer |
12 mL |
4 ℃ |
|
Washing buffer |
30 mL |
4 ℃ |
|
Elution buffer |
800 μL |
4 ℃，Protect from light |
|
Protease inhibitor (100x) |
35 μL |
-20 ℃ |
Special reminder 1:
You need to prepare recombinant protein, PBS, 6X Loading buffer, magnetic rack and other reagents and consumables.
Special reminder 2:
6T is 6 single-group (1 control group or 1 experimental group) pull-down experiments. The following operation steps include 1 control group and 1 experimental group, which requires 2T reagents.
Frequently Asked Questions and Answers
Through WB verification after pull-down, it was found that although the target band was visible, the background was very high:
There are many reasons:
1) The experimental instruments or reagents are contaminated, use clean instruments and reagents.
2) The target-specific adsorption on the transfer membrane leads to high background. Wear gloves during the experimental operation and use tweezers to take it. Do not touch the transfer surface of the membrane.
3) There may be large protein complexes that are not completely dissolved in the prepared sample. After preparing the sample, perform a short ultrasonic treatment (3 times, 5 seconds each time), then centrifuge, and take the supernatant for subsequent experiments.
4) If the washing is not thorough, multiple washings are required, and the NaCl and detergent concentrations in the washing solution are newly increased.
5) If too many cells or tissues are used for lysis, resulting in high background, the sample volume must be reduced. 100-500ug cell lysate is recommended.
6) Protein degradation may also cause high background. Try to use freshly prepared samples.
