Dear all,
I want to purify a final tagging reaction, where a 12kDa protein gets a chemical 1.5kDa tag via enzymatic method by a 60kDa transferase. As it is quite sticky, I don't want to use FPLC SEC & centrifugation, e.g. with Amicon filters.
A neighbouring group has a HPLC and some older Columns, however I am puzzled which one to use:
Vydac protein & peptide C18 (218TP1010)
Vydac protein & peptide C4 (214TP1010)
Vydac protein & peptide C8 (208TP510)
I am aware that too much organic solvents might harm my protein, however there are publications where it is cleaned up after refolding with HPLC (there they use different C18 column we don't have at our hands).
I would go with the C18, but the ones operating the system said the column blocks due to the 60kDa protein, which I don't understand, as this one should simply not enter the pores and elute anyway first, right? Afterwards I suppose to get a nice separation of the 12kDa and the tagged 13.5kDa protein.
best wishes,
Chris
I agree with the comments made by both Jack Silver and William Letter. Most likely you will need a stationary phase with a large pore size. Standard pore sizes of 60 or 100Å will be too narrow. I would also favour a C4 or C8 column since either gives you a wider choice of mobile phases to use. C4 / C8 column can be used in normal mode (= non-aqueous mobile phase) as well as in RP mode (= aqueous mobile phase). Furthermore, running C18 stationary phases perpetually with mobile phases of high ACN content (as may be required so target proteins will actually elute from the column) will over time degrade C18 phases.
You need to check first that the protein is soluble under the studied conditions. Second the size of the pores from the adsorbent has to be large.
You'd probably want a wide-pore column (200 or 300 A). Also, I'd personally choose a C4 since those proteins, not being a small molecule, would only adsorb onto the ends of the alkyl chains and not adsorb between them.
I read the following: " ...ones operating the system said the column blocks due to the 60kDa protein". Is this due to solubility, perhaps? If it precipitates, it would clog the column and not elute.
The refolding issue is a legitimate concern and you'd have to run run a small amount to test this.
There is no way to know for sure without first running a few tests and scouting methods on a larger pore column (300A) to find out which is best.
I agree with the comments made by both Jack Silver and William Letter. Most likely you will need a stationary phase with a large pore size. Standard pore sizes of 60 or 100Å will be too narrow. I would also favour a C4 or C8 column since either gives you a wider choice of mobile phases to use. C4 / C8 column can be used in normal mode (= non-aqueous mobile phase) as well as in RP mode (= aqueous mobile phase). Furthermore, running C18 stationary phases perpetually with mobile phases of high ACN content (as may be required so target proteins will actually elute from the column) will over time degrade C18 phases.
Dear all,
thanks a lot for your answers! I now use a 300A sized C18, with exchangeable safety cartridges to get rid of too big, clogging proteins. Moreover, I use the suggestion by Mr. Meier-Augenstein, and run it usually with max. 70%ACN, and so far I got a nice separation of my proteins (the 12 and 13.5kDa) around 30-40% ACN. As the manufacturer suggested a 95% ACN gradient before storage, I do it once in a while, when putting the column aside, before finally storing it in 65%ACN/H2O. Further I also tested solubility beforehand and did several trial runs with small amounts as proposed.
Best, Chris
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