Isoelectric focusing

Protein extracts from HeLa cells were incubated with alkaline phosphatase (Roche) for 2 h. Each 500-µg protein extract was rehydrated in resolubilization buffer [7 M urea, 2 M thiourea, 2% ASB-14, 0.5% Triton X-100, 1% (v/v) ampholyte, 1% (v/v) tributylphosphine and 0.1% Bromophenol Blue]. Samples were loaded onto the immobilized pH gradient (IPG) strip gels (linear pH gradient 4–7, 7 cm; Bio-Rad) and subjected to isoelectric focusing (Bio-Rad).

2-DE and MS/MS analysis was performed as described previously [54] (link). Briefly, cells were dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.2% pH3–10 ampholyte, Bio-Rad, USA) in presence of protease inhibitor (Sigma). Samples were loaded into IPG strips (17 cm, pH3–10NL, Bio-Rad) using a passive rehydration method, and then subjected to isoelectric focusing (Bio-Rad). The second dimension separation was performed using 12% SDS-PAGE after equilibration. The gels were stained with CBB R-250 (Bio-Rad). Identification and quantitation of protein spots in the gel was achieved using PDQuest software (Bio-Rad).
In-gel protein digestion was performed using mass spectrometry grade trypsin according to the manufacturer’s instructions. The gel spots were destained with 100 mM NH₄HCO₃/50% acetonitrile (ACN) and dehydrated with 100% ACN. The gels were then incubated with trypsin (Promega, V5280), followed by double extraction with 50% ACN/5% trifluoroacetic acid (TFA). The peptide extracts were dried in a speed-VAC concentrator (Thermo), and subjected to mass spectrometric analysis using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source.