GST Pull Down Interacting Proteins

[clokke76]

Hello guys,

I am trying to get specific interactions of two protein using GST pull down assays.

However the problem is that i get a band in the GST control sample.

I did GST pull-down assay to see if my GST-fused protein really binds to another protein in the cell lysate.

• Basic GST Pull Down Protocol
• GST Pull down assay
• Protein elution with Laemmli buffer
• Sds-page
• Western Blotting

I know that BSA can block the non-specific binding of protein to beads.
however i didnt block for feat that the BSA would show up everywhere.

Should I block by using bovine serum albumin (BSA) or another method?

Notes on the GST pull down.
I had pre-cleared the cell lysates with GST and glutathione agarose beads.

How can I eliminate this non-specific bands?

thanks!

[Rebecca27]

Hi,

Did you ever figure this problem out? I am having the same issue and am trying to figure out the best approach to blocking non-specific binding. Any help you could provide would be appreciated.

thanks,
Rebecca27

[Aliya]

I am going to be doing some pull downs too. I will be happy to know how to go around this.

[Moleecule]

Try increasing the concentration of detergent (eg. Triton X-100) in the wash buffer till your negative control shows up to be completely negative.