Hi all! I am currently working on purifying some bioactive peptides. After carefully extracting peptides below the molecular weight of 3 kDa (via ultrafiltration), I subjected them to ion exchange chromatography (IEX) using a weak anion exchanger, and eluted the peptides with 25 mM sodium phosphate, 1 M NaCl (pH 7.5) using linear gradient elution. I have finished freeze-drying (lyophilising) the fractions, but because I didn't perform prior dialysis, I am left with a cotton candy appearing, white solid for all samples, that I'm assuming is salt, and is adding weight to my fractions, especially the acidic ones. My question is, how do I get an idea of the protein concentration in fractions, so I can solubilise them in milliQ water to perform further analysis?
I'm no expert in your area of work, but I can perhaps help a bit with the Lyophilisation part.
Everything that is not volatile, that is to say ALL the salt, buffer salts, and proteins, will be left after lyophilisation. I believe that many protein scientists do dialyse / de salt before drying so that they do not get a salt concentration that may adversely affect the protein. Most folks want to redissolve in a much smaller volume.
The answer to the question you asked, therefore, is that whatever protein you got, and all the salts, will be in the sample after drying.
Side note, watch out for phosphate buffer, I am told it exhibits quite a pH shift on freezing!
Rob Darrington many thanks for your response! I think I am going to go ahead with solubilisation of freeze dried fractions in small volumes (just hoping the salt does not affect the peptide bioactivity).
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