In support of the idea that TRPA1 is activated by electrophiles through modification of these three Cys residues, a mass spectrometric analysis revealed that the irreversible alkylating agent iodoacetamide
modifies Cys415, Cys422 and Cys622 localized in the N-terminal ARD of mouse TRPA1.
WORDS WITH 2 EACH OF 4 DIFFERENT VOWELS iooaeaie IODOACETAMIDE
uaioouia QUASICOLLOQULAL ueuieaia UNSESQUIPEDALIAN ueouioie UNREVOLUTIONIZED auaeoeou AQUAEOLEOUS
Each sample (between 30 [micro]g and 200 [micro]g) was first reduced with dithiothreitol and alkylated with iodoacetamide
and then digested by adding 2 [micro]g sequencing-grade modified trypsin overnight at 37[degrees]C.
8, 2% (m/v) SDS, 20% (v/v) glycerol, 270 mM iodoacetamide
(IAA)], and Anally rinsed with water.
Tenders are invited for Supply Of Consumables For Proteomics, Chaps - Qty : 1 Pack X 10 Mg, Iodoacetamide
- Qty : 1 Pack X 25 Gm
Ammonia bicarbonate (ABC), ammonium persulfate (AP), acetronitrile (ACN), 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS),DNBS, formic acid (FA), iodoacetamide
(IAM), and cell lysis solution were from Sigma (US).
5 M iodoacetamide
was added, and proteins were alkylated at room temperature for 30 min, in the dark.
4 (final concentration of iodoacetamide
was 17 mM), and incubated at 37 oC for half an hour in the dark.
(IAA) and phosphatase inhibitor cocktail were purchased from Sigma (St.
8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT) and subsequently alkylated for 20 min in equilibration buffer containing 5% iodoacetamide
instead of DTT at room temperature.
5% w/v iodoacetamide
(GE Healthcare, USA) instead of DTT.
Samples were reduced with dithiothreitol (30 min at 37[degrees]C), and cysteine sulfhydryls were alkylated with iodoacetamide
(30 min at 37[degrees]C in darkness).