Question

using the knowledge that a lectin can be bound to a support such as an agarose bead, describe how you could isolate the Con-A receptor from the surface of the cell

Answer 1

Isolation of Con-A receptor from a cell surfae includes 4 steps viz lysis of the cell, extraction with aquous phenol, binding of Con-A with lectin bound agarose beads and finally wash and elution.

First the cells were dissolved in the lysis buffer having detergent and extracted with aqueous phenol. The Con-A receptor was present in the aqueous phase of the extract. Then the aqueous phase was incubated with lectin bound agarose bead. The Con-A receptor was first bound to the lectin bound agarose bead. A wash step was performed with 175mM Tris to wash out the non specific binding compounds if any , and finally elution step was performed with 0.1M Melibiose or 0.8M Galactose in 175mM Tris, pH 7.5 to get the affinity purified Con-A receptor.