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
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).