I am attempting to purify a secreted/cell surface protein from a transformed
cell line using lectin-affinity chromatography with lectins specific for
sialic acid. However, no protein appears to be sticking to the columns. The
columns are 0.5.x5cm packed with 1ml MAA/SNA gel (3mg protein). I have tried
loading the samples in DMEM and in PBS, and have eluted either by gravity or
at 0.2ml/min with PBS followed by either 0.1M glycine pH3.5 or 20mM
ethylenediamine. When this failed I attempted to use standard glycoproteins to
check if the columns were OK both fetuin for the MAA and transferrin for the
SNA failed to adhere in significant amounts. My secreted/cell surface protein
is reactive with the lectins after Western blotting using DIG-labelled
lectins in the Boehringer Glycan Differentiation kit. Does anyone have any
ideas as to what the problem may be?
Thanks Ian Mc
