diff --git a/studies/competition co-IP/protocols/ingeldigest.txt b/studies/competition co-IP/protocols/ingeldigest.txt index bc1750e147bbefdcca9675186753fcfc62cce3fd..48a9c39f15983d1adca1a1961d1245c6b27431a6 100644 --- a/studies/competition co-IP/protocols/ingeldigest.txt +++ b/studies/competition co-IP/protocols/ingeldigest.txt @@ -1,2 +1,24 @@ In-gel digest: -1. \ No newline at end of file +1. Run samples at 150 V until the samples run 1 cm into the separating gel +2. 10 min Destainer (40% ethanol, 7% acetic acid); 5 min Destainer; 2x 5 min H2O +3. Colloidal Coomassie 16 h +4. 2x 5 min in 1% acetic acid +5. Cut the bands and cut in 1 mm x1 mm pieces, put in 1.5 mL tube +6. Destain +7. Wash the gel pieces with ~200 µL 40 mM ammonium bicarbonate (ABC) for 5 min (with shaking, 900rpm (should be turbulent shaking!) at ambient temperature. Discard liquid into waste. +8. Incubate gel pieces in 70% acetonitrile (in water) for 15 min (with shaking as before) at ambient temperature. Gel may go cloudy or opaque as it becomes dehydrated. Discard liquid into waste. +9. Repeat steps 2 and 3 two more times (until the gel slice is completely (nearly) colorless). +10. Completely dehydrate gel by adding 100% acetonitrile and let it stand for at least 5 minutes. Discard liquid into waste. +11. Dry gel pieces under vacuum (~5 min). Now you can store them at -20°C + +12. Prepare enough Trypsin to digest all samples. Prepare 10 ng/uL proteomic grade trypsin in 40 mM ABC. +13. Add 30 - 50 μL (depending on the amount of protein) of the enzyme solution from step 12. to each tube. Let tubes stand in ice for 20-30 minutes. Add more 40mM ABC Let sit for another 5 min, then remove excess enzyme solution (if it is really too much). +14. Add 50-100 μL (enough to cover all) 40 mM ABC to each tube and incubate at 37° C for at least 3 h (tubes can be left o/n at this stage). +15. Spin the liquid briefly down and add 2% Formic acid +16. 20 min shaking as before, transfer liquid to a high quality 1.5 mL tube +17. Add 100µl 2%FoAc, 10% ACN to gel pieces, shake 20 min. This extraction will contain the more hydrophilic peptides. +18. Remove the solution and add to the eluate tube. +19. To the gel piece add 200 μL of 60% acetonitrile containing 1% formic acid, shake for >20 min. This wash will contain most of the tryptic peptides. Add this supernatant to the third extraction. +20. Speed vac the samples, first only 25 mbar 20 min, than go down to 2.5 mbar. As soon as the first tubes are “empty†remove them, keep them at 4°C for a short time or -20°C for a longer time. + +