Glycoprotein Stain This procedure is used to stain for glycoproteins (i.e. proteins that have sugar groups covalently attached to them) that have been transferred from a gel to a PVDF membrane. A detailed description of the procedure can be found in the reference given below. Concavalin A (Con A) is used to bind to sugar groups, and then peroxidase (isolated from horseradish) is used to bind to the Con A. A color reaction is used to reveal where glycoproteins are located on the PVDF membrane. This procedure is actually very similar to the immunodetection procedure I described earlier -- there is a series of cross reaction steps alternatin with washing steps, and leads to a color reaction. Again, this is all done with a shaker. Some differences with the glycoprotein stain versus immunodetection are that the glycoprotein stain doesn't use antibodies, and this technique requires less time to carry out. Also, the color reaction occurs within a few seconds to a few minutes (ten minutes at the most). Glycoprotein Stain [ref: Richard Hawkes. Analytical Biochemistry (1982) 123:143-146] Block in TBS-BSA for 1 hour: 50mM Tris-HCl pH 7.5, 200mM NaCl, 2.5% Bovine Serum Albumin (BSA) Shake for 30 min in TBS-BSA containing fresh 50ug/ml ConA Wash 3x10min in TBS (TBS as above without BSA) Shake for 30 min in TBS containing fresh 50ug/ml peroxidase (from horseradish) Wash 4x10min in TBS Detect in fresh: 0.06% 4-chloronaphtol, 0.03% Hydrogen peroxide, in TBS (no shaking is necessary for this step). Rinse in H2O, and store in the dark. Stock Solutions: 3% 4-chloronaphtol in methanol (stored in fridge in the dark) 50ug/ml peroxidase from horseradish in H2O (stored as aliquots in -80 degrees C) 50ug/ml Concavalin A in H2O (stored as aliquots in -80 degrees C) SITE MAP EVOLUTION INDEX. This page hosted by Get your own Free Home Page