How do you generally purify your peptides?
Our peptides are generally purified by preparative reversed phase HPLC, using two tri-fluoro acetic acid (TFA) modified buffers at pH 2. Buffer A is 0.1 % TFA in deionised water and buffer B is 0.1 % TFA in acetonitrile (ACN) at pH 2. Peptides are dissolved in either straight buffer A, or some amount of buffer B then diluted with buffer A. Sometimes it is necessary to use an organic polar solvent like (formic acid or acetic acid) in DMSO or DMF to aid in the dissolving of hydrophobic peptides but this is done on a case-by-case basis depending on the sequence analysis. On rare occasions we can get better solubility and therefore better purification at pH 6.8 so in that case we dissolve the peptide and run the gradient using two alternate buffers. The pH 6.8 buffers we use are 10 mM ammonium acetate in deionised water (buffer A) or ACN (buffer B). The separation is monitored by UV at 214 nm and fractions are collected and analysed by MALDI-TOF mass spectrometry for product identity and by reversed phase analytical HPLC for purity. The fractions are then lyophilized to remove the solvents. The fractions that meet the specifications of the order are then combined into one vial. Next, we run a final MALDI-TOF and analytical HPLC on the combined material.
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10/22/2007