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PROTOCOLS 1

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 MnCl2 (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 4oC.

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

--  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.