PROTOCOLS 1

B:WGAIRS.SAM 7/29/92

WGA-IR PHOSPHORYLATION OF IRS-1^bac

1. Reaction is 60 ul:

-- 10 ul WGA-IR

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

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

-- 30 ul 1X BAC BUFFER (1 M NaCl, 50 mM Tris 7.8)

Begin by adding:

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

(Preincubate 10-15 minutes room temperature to activate IR)

add 5 ul of IRS-1^bac, continue incubation at room temperature for desired time: 3 hr appears to be max, but at low concntrations for Km studies, you should go for 1 minute.

Common variables: --time of incubation.

--concentration of IRS-1 added.

--use of ³²P-ATP not necessary for

some applications, more is desirable

for others.

--reaction can be alterred by omitting

some amount of BAC Buffer, and

replacing it with similar vol IRS-1

when IRS-1 is added.

2. Stop the reaction:

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 not to slop, bubble, or you will fog the gel. Bottom gel buffer is hot waste.

-- 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.