Acceptance for this assay is +/- 5% of reference sample.Į. The observed average measurement of 3 replicate samples was converted to mg/ml using an extinction coefficient of 55,450 and molecular weight of 68,067 Daltons. Protein Concentration (OD 280) MeasurementĪ 3.0 µl sample of enzyme was analyzed at OD 280using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. A8.25-A8.26).Ģ.0 µl of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µl of a 1:100 dilution of the sample. Reactions were incubated 10 minutes at 37☌, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp.
Dilutions of enzyme were made in a glycerol (50%) containing Klenow (3′→5′ exo-) storage solution ( f= 0.12-0.002 µg/µl) and added to 50 µl reactions containing 4 µg Calf Thymus DNA, 1X Blue Buffer, 4m Ci/ml 3H-dTTP and 100 µM dNTPs.
KLENOW FRAGMENT SERIAL
Unit activity was measured using a 2-fold serial dilution method. In 20mM TRIS-HCl, pH 7.5, containing 50% glycerol, 0.1mM EDTA and 1mM DTT. The protein is expressed as a truncated product of the E.coli PolA gene and contains the D355A and E357A mutations. Klenow (3′-5′ exo-) is a mesophilic DNA polymerase deficient in both proofreading (3′-5′) and nick-translation (5′-3′) nuclease activities, and that displays a moderate strand displacement activity during DNA synthesis.
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