Despite the positive load generated by the binding of amine to the activated column of Cyanogen bromide, how can it be justified that the proteins do not stick to the column in a non-specific manner?
Why are you surprised? CNBr was used as a toxic gas in WW1 and bound to many proteins and enzymes to prevent their activity and preserve life.
I am going to use cnbr to active spharose beads for purification of HBsAg and positive charge may lead to bind non specific proteins on column...how can we get ride of this positive charge? I mean we want to purify HBsAg by affinity chromatography and this charge makes it an ionexchange column as well! so in addition of HBsAg ,non specific proteins may bind to column by their charge not by affinity.
Try deactivating the column with an alkaline (OH-) solution before and with your sample. The positive charges on the column will be blocked by the OH-.
My sample is semi purified crude extract of pichia pastoris ,what kind of buffer do you suggest for blocking positive charge somehow that doesnt affect purification?
pH 7.2 Phosphate Buffer (0.10 M Sodium Phosphate and Sodium Hydrogen Phosphate). Maybe Borate Buffer also.
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