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competitive binding


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#1 nicot

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Posted 09 September 2009 - 09:36 AM

I have a compound which mimicking the protein -protein direct binding and enhance apoptosis
I have one protein His tagged and other one with gst tagged. Both are purified
I want to do an assay to show which protein is binding the compound.
I immobilized the gst tagged protein with gst beads then wash then compound then with next interacting protein.
I am not able to see any protein- portien binding in the control or drug treated one .
I found all the interacting proteins are washed off. Can anybody suggest something?

i have done few experiments with incubating gst beads with gst tagged protein overnight, wash then compound, wash then interacting his tagged protein
here I am getting a lot of nonspecific binding of his protein.

#2 BIOKMST

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Posted 17 September 2009 - 11:49 AM

I have a compound which mimicking the protein -protein direct binding and enhance apoptosis
I have one protein His tagged and other one with gst tagged. Both are purified
I want to do an assay to show which protein is binding the compound.
I immobilized the gst tagged protein with gst beads then wash then compound then with next interacting protein.
I am not able to see any protein- portien binding in the control or drug treated one .
I found all the interacting proteins are washed off. Can anybody suggest something?

i have done few experiments with incubating gst beads with gst tagged protein overnight, wash then compound, wash then interacting his tagged protein
here I am getting a lot of nonspecific binding of his protein.


I'm thinking - cross-linking - but, I've never done it and don't know how it can work with lysates....

Good Luck!




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