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GST pull down assay: GST binds to the prey protein. Negative control shows bindi


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#1 Gecko

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Posted 28 May 2013 - 02:24 PM

Hello everyone,

When I did my GST pull down assay, I found GST binds to my prey protein.

My bait protein (GST fusion protein) and prey protein are relatively pure. And this pull down assay contains only bait (GST fusion protein), prey and Glutathione beads. I do not use cell lysate to do the pull down.

My negative control is GST, prey and Glutathione beads.

However I found GST binds to my prey protein strongly.

Do I need to make my binding wash buffer more stringent? My binding wash buffer is only 1xPBS with 1% Tween 20.
Does anyone have other suggestion?

Thanks so much!

#2 bob1

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Posted 29 May 2013 - 07:39 PM

The GST interaction should be quite specific, you can do more washes to remove non-specific binding. Stringency can be improved by raising the salt content.

You could also try blocking the beads with BSA or something similar, or try finding beads made from a different substrate.

#3 Gecko

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Posted 31 May 2013 - 12:05 AM

Hello Thelymitra pulchella,

Thank you for your kind reply.

Could I ask you three questions upon your reply?

1. Pull down assay simulates the protein-protein interaction under physiological condition. If I increase the salt concentration, the whole environment will be quite different from the normal salt concentration in a nomal cell.

2. I did not try to incubate prey protein with Glutathione sepharose beads, previously I just incubated three: GST-bait, prey and beads; or GST, prey and beads.
May be you are right, I can try to incubate only prey and beads and run the WB to test your hypothesis.
You mention BSA, do you use 5%(w/v) BSA like WB process? After block with BSA, you wash it away?

3. You believe GST interaction should be quite specific. You mean GST rarely or never binds to other proteins?

Thank you again for your help!

#4 bob1

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Posted 01 June 2013 - 01:45 AM

1) yes, unfortunately you are right, I was talking about antibody interactions with your target protein when I was thinking of stringency washing. Strong protein:protein interactions will also survive high salt.
2) yep just like WB.
3)lets just say fewer proteins than many other sequences.

#5 Gecko

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Posted 03 June 2013 - 10:52 PM

Hello Bob1,

Thank you so much for your help!




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