IUBio GIL .. BIOSCI/Bionet News .. Biosequences .. Software .. FTP

MAA/SNA Lectins

Steven Pirie-Shepherd srps at galactose.mc.duke.edu
Wed Sep 27 12:14:17 EST 1995


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!"



More information about the Glycosci mailing list

Send comments to us at archive@iubioarchive.bio.net