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ICH2-CONH2; a cerivative of acetamide that reacts readily with sulfhydryl groups and is therefore a strong inhibitor of many enzymes.
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Hydroxyproline, sarcosine, iodoacetamide, and 9-fluorenylmethyl-chloroformate (FMOC) were purchased from Sigma-Aldrich (St Louis, Missouri, USA).
In short, proteins were separated on a NOVEX NuPage 10% gel, proteins were reduced with DTT and subsequently alkylated with iodoacetamide (Sigma).
Iodoacetamide was added to alkylate the proteins and incubated for 1 h in the dark at room temperature (RT).
The strips were incubated for 15 min in 5 mL of equilibrate buffer (6 M ureia, 50 mM Tris-Hcl 1.5 M, pH 8.8, 30% glicerol, 2% SDS, 1% bromophenol blue) containing 50 mg DTT, followed by a second incubation for 15 min in 5 mL of the same buffer containing 125 mg iodoacetamide.
HEPES, sodium chloride, ammonium formate, urea, tris base, iodoacetamide, and tosyl lysyl chloromethyl ketone were purchased from Sigma Aldrich.
After IEF, proteins were reduced and alkylated by soaking the IPG strips in the equilibration solution (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 8.8) containing 1% DTT for 15 min at room temperature and then in equilibration solution containing 4% Iodoacetamide (IAA) for 15 min.
Upon IEF completion, the IPG gel strip was incubated at room temperature for 15 min in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 2% SDS, 30% glycerol, and traces of bromophenol blue) containing 10 mg/mL DTT, followed by a second incubation step (15 min at room temperature) in equilibration buffer containing 25 mg/mL iodoacetamide instead of DTT.
After the strips were subjected to isoelectric focussing, the IPG strip was equilibrated in solution (6 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 0.5% IPG buffer containing 1% DTT w/v for 15 min, then incubated in 4% iodoacetamide w/v for 15 min on Ettan IPGphor Isoelectric Focusing System (GE Amersham).
The gel fragments were rehydrated and reduced by dithiothreitol (DTT) (20 mM DTT/AMBIC 50 mM) at 56 [degrees]C and alkylated for 40 minutes with iodoacetamide (IAA) (55 mM IAA/AMBIC 50 mM) in darkness for 30 minutes.
About 40 mg of protein was dissolved in ammonium bicarbonate 25 mM, reduced with dithiothreitol 5 mM at 80[degrees]C for 30 min, and alkylated using iodoacetamide 10 mM at 37[degrees]C for 20 min.
After isoelectric focusing, the strips were equilibrated in the first equilibration buffer (6 M Urea, 112 mM Tris-HCl (pH 8.8), 30% glycerol, 4% SDS, and 130 mM DTT) for 15 min, followed by the second equilibration buffer containing the same compositions with 135 mM iodoacetamide (IAA) instead of DTT for 15 min.
Acrylamide/bis solution 40%, ReadyStrip[TM] IPG (Immobilized pH gradient) strips 17 cm and pH 47, TEMED (tetramethylenediamine), ammonium persulfate (APS), 2-mercaptoethanol, iodoacetamide, equilibration buffer I and II, mineral oil, overlay agarose, Precision Plus Protein[TM] Dual Color Standards, Precision Plus Protein[TM] Dual Xtra Prestained Protein Standards, Aurum[TM] Serum Protein Mini Kit are from BioRad (Hercules, CA, USA).