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Hydroxylamine Cleavage of Asn-Gly

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  1. 4 M guanidine HCl and hydroxylamine. Buffer to pH 9 with 0.2 M NaCO3. Will need to add ~ 16 volumes of NaCO3 to reach pH 9 since solution is very acidic. Add 5mM DTT, 1mM N-ethylmaleimide and 1x TXSWB to protein solution. Mix equal volumes of guanidine-hydroxylamine solution with your protein. Final protein conc. should be 1-5 mg/ml. Adjust pH to 9 if necessary with NaCO3 Incubate 4-8 hours at 45ºC. Neutralize pH with acetic acid.
  2. TCA precipitate protein.