1 reply to this topic

[nicot]

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.

[BIOKMST]

nicot, on Sep 9 2009, 10:36 AM, said:

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!