DSP nuclear crosslinking

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Fri Apr 29 11:24:18 EST 2011


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, 񟄂რwrote:
> 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
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods



More information about the Methods mailing list