PROTOCOLS 1

B:WGABIND.SAM 7/25/92

¹²⁵I-INSULIN BINDING ON WGA PREP

-- Best done in triplicate.

-- To 3-4 ug WGA purified protein in an eppendorf tube, add 300 ul of binding buffer: H/T containing 150 mM NaCl.

-- to non-specific (control) tubes, add cold insulin to 10^-6 M. (For scatchard analysis, use concentrations varying from 1,2,4,6,8 x 10^-10 M to 10^-7 and 10^-6 M).

-- add 10,000 to 20,000 cpm/tube of ¹²⁵I-insulin (A-14). Bind overnight at 4^oC.

--Add 100 ul/tube 0.4% Bovine gamma globulin, followed by 400 ul/tube 25% polyethylene glycol (PEG). Vortex and incubate 30 minutes at 4^oC.

-- Centrifuge 3' in a microfuge.

-- Remove supernatant, wash twice with 500 ul 12.5% PEG (do not vortex).

-- Spin/remove supernatant

--Drain tubes and count in gamma-counter