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文献和实验Protocols for protein extraction from whole tissues for isoelectric focusing
. 4.Immediately add 4 volumes of ice cold 100% acetone to filtered homogenate,mix by vortexing and place at 20℃for at least one hour to precipitate proteins. 5.Centrifuge at 5000 g for 15 min to collect precipitated protein,decant supernatant. 6.Gently blot
2 EDTA800 mLType I water?/span> 20.5 mL10 N NaOH to pH 7.4x mLType I water1000 mLAutoclave or filter sterilize. SSPE can replace SSC plus NaPO4 , pH 6.7.50% Dextran sulfate MW 5000Dextran sulfate MW 5000 works just as well as the traditional MW 500
. or more. Block in 30 ml of 1X Western buffer (containing 0.1% Tween-20 and 0.2% I-Block), gently rocking, 1 hr, room temperature. Add appropriate dilution of primary antibody (typically 1:5000 or 1:10,000) prepared in 1X Western buffer (containing
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