I have been trying to assess an interaction between a hypothetical protein chromosomally tagged-his and an AMP in vivo (looking at where the protein-AMP interact) pulldown. Because I am asking if the interaction occurs specifically in the periplasm, I can't first add the hypothetical protein and then LL-37 to the beads.
I grow up the bacteria for 3 hr, incubate it with LL-37 (AMP) for 1 hr and then fractionate and/or lyse. After, I incubate the lysate and fractionated portion with preclear beads for 1 hr while blocking the Ni-NTA beads in 5% BSA. After incubation with the Ni-NTA beads for an hr, I wash 7x, elute off, and then boil the elutions. I have been unsuccessful in getting LL-37 to not bind and pulldown with the beads as shown through a western blot and an antibody to LL-37
The binding buffer/wash buffer I use is: .5 M NaCl, .001% Triton-X, 20 mM immidazole and Tris-HCl. Any assistance would be absolutely wonderful
This peptide has lots of positive charge, negative charge, and aromatic groups. High salt should help block ionic interactions. Maybe 0.5M is not high enough. Detergent should help with hydrophobic interactions, but 0.001% Triton X-100 is probably far too low a concentration.
The problem is if the conditions you use to prevent the peptide from binding to the beads also prevent it from binding to its target.
If there are different chemistries of beads that you can use, maybe you can find a type that has less binding to this peptide, but I doubt it. LL-37 looks like it would stick to anything.
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