Hydroxylamine experiments

1. Treat cells as desired, then wash twice with ice-cold PBS.
2. Scrape cells and pellet them at 4 ^oC for 5 min at 300 g. Keep pellets on ice.
3. Lyse cell pellets in SDS lysis buffer (10 mM HEPES pH 8.0, 2 mM MgCl2, 1% SDS, 250 U Universal Nuclease (Pierce; Waltham, MA, US), 1 x protease inhibitor (Roche; Basel, Switzerland).
4. Sonicate manually using a microtip MS 1.5 with 65% amplitude for 30 sec. 
5. Measure protein concentration with BCA assay (Pierce; Waltham, MA, US) according to the manufacturer’s instructions.
6. Prepare the desired amounts of proteins in 2 tubes, one for an untreated control and one for the hydroxylamine treatment.
7. Treat one tube with a final concentration of 1 M NH2OH for 3 hr at room temperature under the fume hood. For example, add 3.3 µl hydroxylamine (50 wt % in H₂O; Sigma-Aldrich; 438227; St. Louis, MO, US) into 50µl lysate. Add the same amount of lysis buffer to the untreated control and incubate them together for 3 hrs.
8. After the treatment, neutralize extracts with the addition of 10 µl of 0.3% HCl and mix with 4x SDS Loading buffer (Invitrogen; Calrsbad, CA, US) containing 100 mM DTT.
9. Proceed with immunoblotting.

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