Dear 尹亚飞 , 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, 尹亚飞 <[email protected]> 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
