Can GST Pull-Down and Western Blot Be Effectively Performed Following Transfection in 293T Cells?

The GST pull-down protocol you described is methodologically sound. It involves the following key steps: transfecting 293T cells with a plasmid encoding a GST-tagged target protein, lysing the cells using an appropriate lysis buffer, purifying the GST fusion protein along with any associated interacting partners using glutathione-conjugated agarose beads, and subsequently analyzing the eluates by Western blot. The detailed workflow is as follows:

 

Transfection of 293t Cells

Introduce a plasmid encoding the GST-tagged target protein into 293T cells using a suitable transfection reagent or method.

 

Cell Lysis

Lyse the transfected cells with a lysis buffer supplemented with protease inhibitors to extract intracellular proteins while preserving potential interactions.

 

Purification of Gst Fusion Protein

Use glutathione agarose beads to isolate the GST-tagged protein from the lysate. Proteins that interact with the GST fusion protein may also co-purify during this step.

 

Elution

Elute the bound GST fusion protein and potential interacting proteins from the beads using a glutathione-containing elution buffer.

 

Western Blot Analysis

Subject the eluted protein samples to Western blot analysis. The GST fusion protein can be detected using an anti-GST antibody, while interacting partners may be identified using specific antibodies targeting the proteins of interest.

 

It is important to note that several experimental conditions—such as the composition of the lysis buffer, the concentration of glutathione in the elution buffer, and the choice of antibodies—may require optimization depending on the nature of the target and interacting proteins.

 

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