Polypeptide mixture isoelectric focusing separation method for proteomics analysis
A technology of proteomics and isoelectric focusing, applied in the field of proteomics, can solve the problems of cumbersome operation, affecting the detection of low-abundance proteins, and poor reproducibility of experimental results
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[0027] The following reagents, acetonitrile, water, and formic acid, were all chromatographically pure and were purchased from Sigma-Aldrich Reagent Company; mass spectrometry-grade trypsin was purchased from Promega Company; dry strips and sample cups were purchased from Bio-Rad Company; the mass spectrometer was a linear ion Trap-Electrostatic Field Orbitrap Combined Mass Spectrometer (Thermo LTQ-Orbitrap Elite).
[0028] (1) After ultrasonic crushing of buffalo sperm, add lysate (7M urea, 2% CHAPS, 0.5% DTT and 0.5% PMSF) to digest and extract total protein, centrifuge to remove cell debris and other impurities, and discard the lower layer of cell debris;
[0029] (2) Take 500 μL of the supernatant extracted above, add 1.5 mL of pre-cooled acetone to precipitate overnight, centrifuge at 12,000 rpm for 1 h, discard the supernatant, and wash the precipitate with 100 μL of 40 mM ammonium bicarbonate (NH 4 HCO 3 ) reconstitution;
[0030] (3) Add 10 μL 40 mM dithiothreitol (D...
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