PROBING SH2 DOMAIN-CONTAINING

PROTEINS WITH 32P-IRS-1

 

A.  IMMOBILIZE SH2 PROTEINS ON NITROCELLULOSE (DAY 1):

 

1.  Run 1 mg of SH2 domain fusion proteins per lane in a Bio-Rad miniprotein apparatus with 10 well comb.  15% gel.  Use low range markers, run off dye front.  Negative control is GST alone (or similar); GST runs about 29 kDa, so don't run off the 27 kDa marker.

 

2.  Transfer gels to nitrocellulose in Towbin +SDS +MeOH.  40-60 mA O/N or several hours at 100 mA in Bio-Rad mini Transblot.

 

3.  Rinse membranes 2x in dH2O, wash 4 times 10 minutes in washo I:

                                                                                                                (500 ml)

                                                10 mM Tris 7.4                          5 ml 1 M stock

                                                200 mM NaCl                                            20 ml 5 M stock

                                                0.05% Tween                                             0.25 ml

                                                [FRESH b-ME                           70 ml/100 ml]

 

4.  Block membranes overnight at 4o C in FRESH blocko:

                                                                                    (100 ml)

                                    10 mM Tris 7.4                        1 ml 1 M stock

                                    200 mM NaCl                          4 ml 5 M stock

                                    5% NF Dry Milk                      5 g

                                    b-ME                                      70 ml

 

B.  PREPARE PROBE AND PROBE MEMBRANES (DAY2):

 

1.  Set up one reaction for each probe.  Each reaction:

                                                            WGA-IR                      30 ml

                                                            100 mM MnCl2           7.2

                                                            2X BAC                      40

                                                            insulin                            1

                                                            1 mM ATP                   4.8

                                                [[INCUBATE 20' RT]]

                                                            HOT ATP                    32

                                                            Crude IRS-1                1-4 ml

                                                [[incubate 2 hr RT]]

 

2.  Remove 2 ml of each reaction; laemmli-ize and run on minigel to check incorporation while completing probe preparation.

 

3.  Add 360 ml/tube of DTT/Guanidine solution:  [4 ml 8 M Guanidine + 28 mg DTT].  Incubate 5 hours at 55o C.

 

4.  Add 25 ml/tube of IDA/Guanidine solution:  [23 mg Iodoacetamide in 75 ml 8 M Guanidine].  Incubate 20' RT in the dark.  While this is incubating prepare 0.5X BAC with 1% BSA, and incubate 1.5 ml of this in Centricon-30 concentrators (30-60 minutes).

 

5.  Add 5 ml/tube of b-ME, incubate 5-10 minutes at room temperature.

 

6.  Add entirety of each reaction tube to a blocked Centricon (from above) and spin at 5000 rpm in JA-17 rotor in Beckman superspeed centrifuge.  (Covering the top of the centricon with parafilm reduces contamination of rotor/centrifuge).  Wash 2-3 times (Refill with 1 ml 0.5X BAC and recentrifuge).

 

7.  Invert centricons into collection tubes and spin briefly.  Add probe to 10 ml blocko and probe membrane overnight at 4o C, rocking.

 

C.  WASH MEMBRANES (DAY 3):

 

1.  Remove probe and save (4o C).  Wash membranes extensively (6+ times) with Washo II:

                                                                                                (500 ml)

                                                10 mM Tris 7.4                        5 ml 1 M stock

                                                200 mM NaCl                          20 ml 5 M stock

                                                0.01% Tween 20                      50 ml

                                                [FRESH b-ME                        70 ml/100 ml]

 

2.  Continue washing membrane with washo II until no counts are eluting from the membrane with subsequent washes.  Dry membrane and expose.