I'm trying to biotinylate surface membrane proteins with NHS-LC-Biotin with Strep-magnetic beads. I'm having trouble with identifying membrane protein with MS (very few identified protein and only 10% are membrane protein of all identified protein) But I find many before using NHS-SS-biotin instead of NHS-LC-biotin. I understand that SS can be broken with reducing agent and LC cannot.
Is there a different between elution of protein-NHS-S/S-biotin using DTT and elution of protein-NHS-LC-SS-biotin using boiling?
Which method would be better for identification of surface membrane protein in MS after SDS-PAGE?
Hello Sugli, hope all's well.
One of the main differences between these biotin reagents (as you know) is that when you add DTT to the NHS-SS biotin, the linkage is cleaved at the disulfide bond and you are left with a smaller moiety on the protein than the equivalent NHS-LC (which does not have a disulfide bond). Depending on the location of this moiety will strongly influence the quality of the quality of the protein digest.
In addition, do you know how well you are biotinylating your membrane proteins? We assume the surface is very crowded and so some people use PNGase F enzyme in an effort to improve biotinylation.
Also, the effective disruption and solubilisation of membrane proteins will play a big role in your identification of surface membrane proteins.
I would use the NHS-SS biotin where possible as the addition of DTT will assist in the denaturation of membrane proteins downstream.
Good luck,
Peter
In our lab, we have always found biotinylation reagents problematic. Also, you might want to be very careful with elution by using boiling, as it may affect the native structure of proteins. We use magnetic nanoparticles loaded with NHS groups in our lab. See the attached poster for more info.
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