1. To your sample, add Super E, protease inhibitors, 1 mM cold Met, antibody.
TMT extract or medium - dilute with Super E to convenient volume; add protease inhibitors, cold Met and antibody.
Some antibodies work only after the antigen has been denatured (e.g. Ab 877, Ab 471): to 50 ul of sample in TMT, add 5 µl 10% SDS and incubate at 100 oC for 5 min; allow to cool to room temperature for 5 min; add IN THIS ORDER: 25 µl 15% NP-40, 180 µl Super E, 3 µl PMSF, 3 µl inhibitor mix, 10 µl antibody. Super E: 50 mm phosphate, 1% Triton.
2. Allow antibody to bind overnight at 4 oC (4 h is sufficient for
3. Spin samples 3 to 5 min in hot microfuge at top speed.
4. While samples are spinning, number a new set of tubes; add as much Super E as you can (at least 0.5 ml) and 60 µl Protein A agarose slurry (1:3) for 10 µl antiserum.
5. Transfer supernatant to new tube with Protein A; do not be greedy and take every last bit of supernatant - you will get dirty IPTs.
6. Shake 60-90 min at room temperature; do not go much longer than this.
7. Wash beads: spin 10 sec in hot microfuge - wash 3 X in SuperE and 2 X in PB. Invert tubes to resuspend beads for each wash.
8. Resuspend beads in SDS samples buffer (60 µl for slab gels; 105 µl for tube gels) with blue or pink tracker dye - DO NOT PUT HOT PIPETTES INTO 15 ml TUBES - YOU WILL END UP WITH HOT SAMPLE BUFFER.
9. Immediately (not a few minutes later) boil samples for 5 min.
10. Allow to come to room temperature; spin to collect liquid.
11. Count aliquot (2 or 5 µl).
12. Load gels: slab gels: 50 mV overnight - leave a note on board if you want gels checked in am
180 mV maximum during day- 4-5 h
tube gels: 20 mV overnight
50 mV maximum during day
13. Slab gels - 30 min in preamplify; 30 min in Amplify/Enhance
14. Dry gel - check/empty trap before you dry gel; 90 min at 70 oC is usually enough.
15. Tube gels - elute slices overnight in SDS gel elution buffer; squirt; count