Ph 3 10nl gel strips

Platelets from patients with myocardial infarction and non-ischemic chest pain were lysed in 100 µL of CHAPS lysis buffer (8M Urea, 4% CHAPS, 2% DTT) and purified using a 2D Clean-up kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions. Comparative 2D gel analysis of the proteomes was performed as described previously with slight modifications [34 (link)]. First dimension isoelectric focusing was performed using a Protean IEF Cell focusing unit (BioRad, Hercules, CA, USA) with pH 3–10NL gel strips (11 cm, GE Healthcare, for some subsequent gels pH 4–7). For the second dimension, equilibrated (5.7M Dithiothreitol and 1.5M Iodoacetamide) gel strips were applied to 12% polyacrylamide gels. Proteins were stained with Lava purple fluorescent staining (Fluorotechnics, Sydney, Australia). Protein spots of interest were excised manually from the gels, digested with trypsin and analyzed by LC ESI-MS/MS as described previously [35 (link)]. Peptide sequence analysis by mass spectrometry of the isolated protein spots resulted in the identification of the differential cleavage of kindlin-3 in the myocardial infarction group.

Platelets from patients with myocardial infarction and nonischemic chest pain were lysed in 100 µL of CHAPS lysis buffer (8 M Urea, 4% CHAPS, and 2% DTT) and purified using a 2D clean-up kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions. Comparative 2D gel analysis of the proteomes was performed as described previously with slight modifications [34 (link),35 (link)]. First dimension isoelectric focusing was performed using a Protean IEF Cell focusing unit (BioRad, Hercules, CA, USA) with pH 3–10 NL gel strips (11 cm, GE Healthcare, for some subsequent gels pH 4–7). For the second dimension equilibrated (5.7 M Dithiotreitol and 1.5 M Iodoacetamide) gel strips were applied to 12% polyacrylamide gels. Proteins were stained with Lava purple fluorescent staining (Fluorotechnics, Sydney, Australia). Protein spots of interest were excised manually from the gels, digested with trypsin. and analyzed by LC ESI-MS/MS (Applied Biosystems/MDS Sciex, Darmstadt, Germany) as described previously [35 (link)].