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.