DSP nuclear crosslinking

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Fri Apr 29 16:12:19 EST 2011


Dear 	=D2=FC=D1=C7=B7=C9 ,

I have a general question first: Is there any chance that a
crosslinker makes it all the way towards the nucleus? There are so
many molecules with amino groups on it's way while it passes the cell
membrane and travels through the cytosol. In my experience with NHS
esters and the labeling of antibodies, the reaction is quite fast. Or
do you isolate the nuclei before you add the crosslinker? NHS esters
also require slightly alkaline pH ( I do my labelings at a pH between
8 and 9). Any added amines (like eg tris, amino acids etc) will quench
all your reagent before it can label any complex.
Do you have a proven reference for your method and working positive
crosslinking control? Is your reagent ok? It's not stable as a
solution; freezing in ultrapure, really dry DMSO will do for some
weeks, however.

Is there a special need for DSP? DSS (no cleavable -SS- bridge) might
be more convenient, as there is no risk of breaking the crosslinked
complex by accident (as inside the cell, the climate is reducing)

You also might try a bis-maleimide analogue of the reagent which will
react with cysteines instead, if N-modification renders your protein
undetectable (as you write you can't detect even the monomers after
adding the reagent). Maleimides work at neutral pH. At alkaline pH,
they also react with amino groups.

Another option could be photoactivable crosslinkers, as you first may
incubate the nuclei (or even cells) until the crosslinker is evenly
distributed before you start the reaction.  Likewise, you also might
perform a pre-incubation on ice before you accelerate the reaction by
warming when using DSP/DSS etc.

If you're working with mammalian cells, a way to isolate the nuclei is
to lyse membranes and remove the cytosol with ice cold PBS/1% TX100.
Then wash with PBS (to remove any detergent), then do the cross-
linking, wash away excessive reagent with PBS, then lyse your cells
with SDS or mechanical force (ultrasound, high speed shearing mixer
etc.).

Finally, you also might need to vary incubation time, temperature and
reagent concentration. And spacer length.

Good luck!

Wo

On Apr 29, 5:02 am, =D2=FC=D1=C7=B7=C9 <tuzi8780... from gmail.com> wrote:
> I'm a student of Tsinghua University, I'm doing a crosslinking IP recentl=
y,
> 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 buffe=
r
> you use? Thank you!!!



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