HELP, I'M LOST IN LECTIN-LAND!

andrew weiskopf aweiskop at lynx.dac.neu.edu
Mon Sep 16 16:04:50 EST 1996


Dale Karlson (dtk at omni.cc.purdue.edu) wrote:
: To whom it may interest,
: 	I am currently attempting to use lectins to qualify sugar residues on a
: glycoprotein of interest which is fixed to a nitrocellulose membrane.  Color
: reactions have been extremely faint.  I am using a concentration of 5 ug/mL;
: is this appropriate?  Experimental concentrations in literature have been very
: vague;  likewise the necessity of salt usage.

Many lectins require metal ions for their activity. I perform binding
steps of a similar assay in Tris-saline buffer (pH 7.5), with 1mM of MgCl2,
MnCl2, and CaCl2. Check your literature to see if other metal salts are 
necessary.

: 	The peroxidase labelled lectins that I am using are Ulex europaeus (UEA 
:  1), Triticum vulgaris (WGA), Glycine MAX (Soybean), and Ricinus communis (RCA
: 120).  Do you know of specific proteins that could be used as positive controls
: on my blot to gauge reaction time/intensity etc.?

Of the ones you've named, I've only worked with WGA, and I got a rather
faint response using a digoxigenin-labelled system (Boehringer
Mannheim). I have found that Galanthus nivalis (GNA) gives a very clean
and sensitive response for oligomannosyl-bound proteins, and sambucus
nigra (SNA) is suitable for detecting many hybrid or complex
oligosaccharides. 

Controls: For GNA, use carboxypeptidase Y, and for SNA, try transferrin.

Good luck


: 	I appreciate any advice that you may provide for my questions.

:                                                        Sincerely,
:                                                    Dale T. Karlson

Andrew Weiskopf
The Barnett Institute
Northeastern University
aweiskop at lynx.dac.neu.edu



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