Hi all, I'm trying to set up a protein-protein binding assay using formaldehyde (FA) crossslinking. I used formaldehyde before for RNA-protein binding experiments so I've pretty much reproduced those experiment conditions, but I haven't had the expected results, not even in the possitive control. What I've done is incubate both proteins in binding buffer (HEPES, EDTA, glycerol, pH 7.9) for 30 min at 37ºC, crosslink for 15 min at RT with 1% FA, stop the reaction with 0.25M Glycine for 5 min at RT, add laemli loading buffer (with B-mercaptoethanol) and load onto a 10% denaturing gel, then fix the gel, dry and expose it o/n. One of the proteins is radioactively labelled and both have been translated in vitro using Promega's TNT system. what I expected to see was a shift in the band size of the labelled protein that had been incubated with the second protein compared to the labelled protein alone. This is the first time I use this protocol to assess binding between two proteins so does any of you have a crosslinking protocol that works (since mine doesn't)?. Many many thanks for your help!
Amparo
FA might not be the best solution for your Xlinking experiment. The Xlinking might have been reverted when you boiled your sample in laemli. You should try another Xlinker like a NHS-ester Xlinker. Plus they work in vivo. Have a look at http://www.piercenet.com/products/browse.cfm?fldID=4CE9DCFE-5056-8A76-4EAC-FCDAAEABC855
thank you so much! I'm giving FA another go just now, if it doesn't work I'll go for your suggestion, thanks!
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