Hi, Could you please provide more details on your protocol? Do you add the crosslinker before or after the biochemical fractionation? Do you lyse the nuclei before adding DSP?
Crosslinkers like DSP react with amine groups on the N-termini of proteins and on the side chains of some amino acids. It is possible that the DSP is reacting with some of the residues that are required for your antibody to recognize the antigen. Have you tried using a different antibody? DSP could also cross-link your protein of interest to other proteins, forming a high-molecular-weight complex. If the complex is not too big, it might run at a different molecular weight than you are expecting for your protein of interest. If the complex is too big, it could be trapped in a pellet if you centrifuge your samples before loading on the gel, or it could be excluded from the gel and trapped in the wells. Luckily, DSP can be cleaved with reducing agents like beta-mercaptoethanol or DTT. Do you add these to your sample loading buffer before loading onto the gel? Hope this helps, Irit On 4/28/2011 8:02 PM, 尹亚飞 wrote: > I'm a student of Tsinghua University, I'm doing a crosslinking IP recently, > and the crosslinking regent is DSP. My target protein complex is in the > nucleus, however, during my experiment, I found my target protein of the > crosslinked sample even could not be detected in nuclear exact, while the > control sample(i.e., sample without crosslinking) performed well. I tried > different lysis buffer from High Salt solution(350mM NaCl) to RIPA > buffer(0.1%SDS, 1% Triton X-100), so I do wonder what kind of lysis buffer > you use? Thank you!!! > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
