I am using size exclusion chromatography to purify a protein. I made a mistake of leaving the pump on and the buffer solution ran dry and made the column run dry. I removed the air and now the column looks uniform. After injection of aceton I got no peak at al. but when I inject the protein, the peak is in the right place.
why do you think this happens? Do I need to repack it?
Dear Hakimeh,
Introducing air in your column you can completely destroy it performance. Columns packed with soft media, like superdex, especially sensitive to air. However, you should check the efficiency and peak shape of your restored column first. If the values are close to those reported by supplier - you are happy. Use acetone or B12 vitamine to determine asymmetry and number of theoretical plates. Instructions can be found in GE Healthcare. Then run a set of protein standards ( RNase A, Carbonic anhydrase, BSA etc.) similar to those supplied by GE Healthcare.
Is this manually packed column? if yes then unpack it and repack it. Do column efficiency test with Acetone. Calculate asymmetry and HETP. If it is matching with your new column performance then try with your sample and check for the performance. if it is ok then you can use it for your purification.
You can also make calibration curve with protein standard as per mentioned above.
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