competitive binding - binding problem (Sep/09/2009 )
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.
nicot on Sep 9 2009, 10:36 AM said:
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!