Hello!
I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and PMSF). Is too much salt, for sephacryl S-100 resin?
Does somebody use an open column?
P.S: After that, I'll run a RP-chromatography
Hi Cristiane,
As Adam already has answered the salt concentration is fine. But I wonder what you mean with "open column"? Is is that you don't have a top adapter and not the possibility to use a pump to load the sample and then run the buffer? You say that you are using Sephacryl S-100. This resin has bead size of 90 um and to get a good separation you should use a column bed with approx. 60 cm bed height and that will be very difficult to run using gravity flow. Perhaps I have misunderstood you?
Another question...why are you starting with SEC this is most of the time the last polishing step when doing purifications due to the small sample volume you can load to get good separation. Why not do a quick desalting after your lysis of the cells and instead go for IEX? If you give me more details about you sample, sample volume and if you know anything about the concentration of your target peptide I can hopefully give you some more detailed suggestions.
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