PROTOCOLS 1

B:WGAAUTO.SAM 7/25/92

WGA-IR AUTOPHOSPHORYLATION

1. Reaction is 50 ul:

-- 3-4 ug (10-20 ul) WGA-IR

-- 2.5 ul MnCl_{2 (100 mM)}

_{-- 5 ul 10}^-6 M insulin or water

-->volume to 45 ul with 50 mM Hepes pH 7.4 containing 0.1% Triton X-100 (H/T)

(Preincubate 15' room temp to allow insulin binding)

-- 5 ul 500 uM ATP containing 20-30 uCi g-[³²P]-ATP.

(Incubate 20 minutes room temperature)

Common variables: insulin concentration,

ATP concentration

time of incubation

2. Stop the reaction (two methods):

a) Laemmli-ize the sample and load directly

-- add 50 ul of 2x Laemmli Sample buffer to each reaction. Close the tube, use a 21 ga needle to put a hole in the lid and boil in water bath for 5 minutes. Load to gel (7.5% SDS-PAGE), careful no to slop, bubble, or you will fog the gel. Bottom gel buffer is hot waste.

b) Immunoprecipitate the sample: 50 mls:

-- add 500 ul of IP buffer: H/T containing

2 mM Vanadate 18 mg

NaF 210 mg

2 mM PMSF 500 ul of stock

5 mM EDTA 500 ul of 0.5 M

-- add 20 ul/reaction of anti-PY or anti-IR antibodies, allow to bind 2 hrs-overnight a 4^oC.

-- add 100 ul/reaction of pansorbin. > 1 hour 4^oC.

-- spin down 2 minutes in microfuge, aspirate supernatant (hot waste). Add 1 ml Tris-SDS IP wash, sonicate. Repeat 2 additional times. Aspirate supernatant, add 70 ul of laemmli sample buffer.

-- boil 5', vortex to resuspend pellet, boil 3'. Spin down pansorbin 5', load supernatant to gel.