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sodium dodecyl sulfate-solubilized proteins

Time:2015/11/28 5:50:06

Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

The sensitivity with which RNase and DNase activity can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) varies widely, depending upon the particular SDS preparation used for electrophoresis. (See also S. A. Lacks, S. S. Springhorn, and A. L. Rosenthal, 1979, Anal. Biochem.100, 357–363.) Sensitivity of detection is greatly increased by using buffered 25% isopropanol, rather than buffer alone, to wash detergent from gels after electrophoresis. Thus it is routinely possible to detect bovine pancreatic RNase A at the picogram level. Use of isopropanol improved activity staining of RNases with each of the 10 SDS preparations examined, including one containing 32% tetradecyl sulfate and 4% hexadecyl sulfate, and reduced the variability from preparation to preparation observed when buffer alone was used to remove SDS. Other water-organic cosolvent binary mixtures can be used but none shows advantages over aqueous isopropanol when sensitivity of detection as well as availability and cost of organic solvent are considered.

A linear Lowry-Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins

The protein method of Lowry, Rosebrough, Farr and Randall was modified to give a linear standard curve of absorbance versus μg of bovine serum albumin at 650 and 750 nm wavelengths (1 cm optical path) over the range up to 50 μg protein per ml and an absorbance of 1.1. This was achieved mainly by using a high concentration of Folin-phenol reagent, added rapidly in a volume that was large relative to the final volume. Sodium dodecyl sulfate (SDS) incorporated in the test did not change the albumin standard curve, and linear results were obtained with water-insoluble membrane proteins (myelin proteolipid and rhodopsin) solubilized by SDS.