deisme - 20-10-2007 at 10:33
I am doing an Experiment about Chromatography. But there are some pre-questionaries that needed to be answered. I wonder if anyone can help me out ?
Q :
Admiting that you have made the following mistakes during the purification of lysozomes (isoelectric point=10.7, Mr=14300) in a chromatography of
molecular exclusion using the gel Sephadex G-100. What are the expected consequences to the final results of the chromatography if..
* During the application of the sample you let partially dry the column of Sephadex G-100 ?
Thank you so much!
chemoleo - 20-10-2007 at 17:06
Probably none at all, as lysozyme is quite stable. Just elute with more buffer, but of course expect the protein to come out later as air won't carry
the protein forward in the column.
However, being a protein, air exposure may cause precipitation on that very gel filtration column. To remove the air, wash the column with 50% MeOH,
about a litre. Then remove the precipitated protein (which is potentially stuck to the column) with 6M guanidinium hydrochlorid. Your column should
then be fully regenerated....