I am trying to enrich for an unstable, potential protein-protein interaction. I've read several papers that have used the chemical crosslinker, DSP. Has anyone successfully used this reagent? If so, might they have some tips and possibly a protocol on how to correctly use it? Concentration, time, etc. Thanks so much!!!
NHS-esters reactions are fast and amine-specific. Because lysine residues are abundant and easily accessible of on the hydrophilic surface of most proteins, they usually crosslink with high efficiency. Using homobifunctional linkers is a "shotgun" approach to capturing interaction complexes; the reagents will crosslink any and all interacting molecules whose respective lysine residues come within the spacer length of the crosslinker. All that is needed to detect a particular interaction complex after crosslinking and (usually) cell lysis is a specific antibody or other probe for one of the target molecules in the complex.The new diazirine photo-reactive linkers combine robust NHS-ester chemistry with a light-activatable reactive group that is able to crosslink with molecules where no amine residue is available or accessible (even DNA, polysaccharides and other molecules). Diazirines are more efficient than traditional photo-reactive moieties (aryl-azides) and give higher yields. These heterobifunctional linkers enable “two-step” reactions in which "bait" proteins can be labeled, added to a cell and light-activated to crosslink at the desired time (e.g., upon cell stimulation when the interaction of interested is presumed to occur.
This is a blast from my past. 20 years ago during my PhD stuies I spent ages trying to get DSP and a variety of crosslinkers with different spacer lengths to work crosslinking photosystem II - to no end. I think you need a serious element of luck that the Lys resdes are clustered in the right range.
It's worth considering EDC as an alternative if you have amine and Caboxyl groups close to each other - it's a zero length peptide bond catalyser. It's the same blunderbuss approach though.
Good luck.
I have recently developed a method to detect hard to extract transmembrane protein-protein interaction using DSP crosslinker. It has worked well because we confirmed the interaction that we got from crosslinker using in vitro pull down assay. You can check my paper and follow or modify the protocol. The method is applicable to study any protein-protein interaction in live cells. You can contact me, if you need more details.
Article MCLIP, an effective method to detect interactions of transme...
Hi Mohammed,
Could you give more details about how to perform the crosslinking reaction? You did this ) in standard culture media? DSP is water-insoluble and it should be dissolved first in DMF or DMSO, right?
Thanks.
Hi Congcong,
Yes, it needs to be first dissolved in a water miscible solvent, such as DMSO or DMF, when the reaction takes place in an aqueous solution. The protocol for ProteoChem's DSP can be found here:
www.proteochem.com/dspcrosslinker100mg-p-96.html
http://www.proteochem.com/dspcrosslinker100mg-p-96.html
Be sure to use tissue culture-grade DMSO and to open a fresh vial for these experiments. This is especially important if you plan to add the crosslinker to living cells because opened DMSO picks up enough water from the atmosphere to react with the cross linker. Also, DMSO oxidizes, and your cells will get sick from the oxidation products or other contaminants present in that old bottle of DMSO in your fume hood.
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