Preserve the activity of the agarose-bound lectins for repeated use
Elution in neutral pH range
All components can be subsequently removed by dialysis
Glycobiology, Affinity Chromatography
1)Wash lectin agarose column to remove unbound proteins with HEPES- or TRIS-Buffered saline (HBS, TBS), pH 7.5, until OD280nm reaches background level.
2)Apply Glycoprotein Eluting Solution and allow to flow through the column by gravity.Note: Do not apply pressure to speed elution because this may reduce the percent recovery of the bound glycoprotein.Complete elution may take up to 10 column volumes.*
3) Following elution, the agarose lectin column is ready for reuse after equilibrating with 2-4 column volumes of HBS or TBS, pH 7.5.
*These results may not be typical for other glycoproteins, since oligosaccharide structures of glycoproteins vary significantly.The eluting solution has a high osmolarity.If downstream applications are adversely affected by salt, eluted glycoproteins may require dialysis or gel filtration.
What is the primary composition of the Vector Labs glycoprotein eluting solutions? Do any of these contain protein?
The glycoprotein eluting solutions are largely comprised of specific sugars and defined salts that are usually required to remove bound material from agarose conjugated lectins. These eluting solutions do not introduce harsh pH conditions or other factors that may affect the eluted glycoproteins or the lectin column. As such, we view these mixtures as being both eluting and regeneration solutions. None of these solutions contains protein.
Glycoproteins are frequently isolated and purified from protein mixtures using columns of agarose-bound lectins. After applying a protein mixture, the agarose-lectin column is washed free of unwanted proteins and the glycoprotein bound to the lectin is eluted with a sugar that inhibits binding. Unfortunately, achieving complete elution with a simple sugar solution can be difficult. Vector Laboratories has developed five Glycoprotein Elution Solutions in the neutral pH range that maximize the yield of eluted glycoproteins and preserve the activity of the agarose-bound lectins for repeated use. All components of these ready-to-use Glycoprotein Eluting Solutions can be subsequently removed by dialysis. These Glycoprotein Eluting Solutions offer researchers convenience and superior recovery over standard sugar solutions.