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文献和实验by reaction with alkaline phosphatase-conjugated goat anti-mouse secondary antibody. Choose the actin pool broadly, however, consider also checking the column fractions by dot blot with mAB 3F2 to identify capping protein. Pool the actin fractions
LABELING MUSCLE ACTIN WITH IATR
,small vial. Procedure (perform under reduced light,4℃ unless otherwise noted) 1.Resuspend 10 mg lyophilized actin in 2 ml buffer 1.Be careful not to make bubbles. 2.Add DTT (100 mM stock)to 5 mM. 3.Dialyze against 250 ml buffer 1 overnight
Labeling Muscle Actin with 5-iodoacetamidofluorescein
with the dialysis buffer and read the OD at 495 nm. D/P = {OD495 x 41 / 60,000} / {(mg/ml)/ 43,000},should be 0.5-0.9. 3.Dilute to 3-5 mg/ml with the dialysis buffer.Calculate total mg of actin.Store as aliquots in liquid N2 after dissolving 2 mg sucrose per mg
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