Ian McFarlane (imcfarlane at ninewells.dundee.ac.uk) wrote:
: 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
--
I run SNA columns in 50mm tris/1mM MgCl2/1mM CaCl2. the metals are
crucial. I elute using 0.1M Lactose in the buffer above. Lactose works
with SNA, I am not sure with MAA. Remember that SNA is a(2-6) specific
AND MAA is a(2-3) specific. I dont think PSA [a(2-8)] will bind to these
lectins. But it sound to me as if you are having problems binding. Use
the metals.
__________________________________________________
Steven Pirie-Shepherd
srps at galactose.mc.duke.edu
"Insert your own pithy phrase just about here!"
