Reversed-Phase chromatography for protein purification - (Jun/13/2012 )
I am new here. I have a question for all biochemists and analytical chemists out there.
We are trying to purify different isoforms of a protein that different very slightly in methylation and charge, and in the past it was done by using RPC-5 columns (reversed phase-5 chormatography columns). We're talking about late 70s here.
So first of all, has anyone ever heard of/had experience with these columns?
Is there something currently available out there that works in a similar way? We've been trying to look for any manufacturer that still makes RPC-5 columns, or something better/similar.
Please help. This is something we've been stuck on in the lab for a while and its becoming a huge bottleneck in moving our experiments forward. Standard HPLC, sedimentation, ultracentrifugations have all only given us partially purified forms of the protein, and we've been unable to get the pure protein!
Any information will be helpful at this point. Thank you so much!
searching the literature i found that rpc-5 is prepared by coating plaskon ctfe-2300 with methyltrialkyl (C8-C10) ammonium chloride (adogen 464).
plaskon ctfe-2300 (poly(chlorotrifluoroethylene)) is no longer produced (it was from allied chemical which has since become part of honeywell). it had been replaced by plaskon ctfe-2100 but that didn't work as well (and plaskon doesn't show up in their catalog at all, anymore)
sigma-aldrich sells poly(chlorotrifluoroethylene) powder but it doesn't give the subtype (ie-2300). you could try it but it may or may not work well. they also sell adogen 464.
Thank you! Looks like there are some other chemicals with the same CAS number out there. I'm thinking of trying those, along with the ones you suggested. Thanks!!
Are you still interested in RPC-5 columns?
I randomly came accross your message today on the web although this is not currently my field. I made RPC-5 columns during my PhD to purify modified tRNAs back in 1995. I have the method written up in my thesis if you want more details.