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Researchers have devised a method to determine SDS concentration in a protein sample that can easily be employed to determine the concentration of SDS in a protein sample during the purification process or other biochemical experiments.
Sodium dodecyl sulfate is an anionic detergent that is most commonly used in the molecular biology and biochemistry. However, its application is limited because it is hard to remove from the protein sample once added to the sample. Though there are some quantitative methods for the determination of SDS concentration in a protein solution but only a few of them are practical and precise. One of such methods is the separation of SDS using an organic solvent. However, this method is tedious and time-consuming.
Therefore, researchers have devised a spectroscopic method to determine SDS concentration in a protein sample. This method uses a dye called as Stain-All that can detect SDS concentration ranging from 0.001-0.01 %. Matrix components such as proteins and nucleic acids can also bind to the dye, Stain-All but they have no effect on absorption (at 453 nm) contributed by SDS bound to the dye. The researchers have optimized this procedure to obtain an accurate and reproducible result.
- Prepare 50 ml of 2 mg/ml stock solution of Stain-All dye in N,N-dimethylformamide (DMF) and store the reagent at 2-8 ºC in a dark bottle. During each use, dilute the stock solution of the reagent using HPLC grade water in a proportion of 1:20 v/v.
- Prepare 50 ml of 1 % stock solution SDS in HPLC grad water. From the stock solution, prepare six standards of SDS solution with the following concentrations; 0, 0.001, 0.0025, 0.005, 0.0075, and 0.01 %.
- Use 0.0033 and 0.0066 % SDS solutions as assay controls for each analysis and in a similar way, use 1.1 and 2.7 mg/ml of recombinant protein solutions with different SDS concentration as the sample controls.
Spectrophotometric quantification of SDS
Spectrophotometric method to determine SDS concentration of a protein sample can carried out by plotting standard calibration curve of SDS. 1 ml of assay mixture containing 640 µl of HPLC grade water, 100 µl of phosphate buffered saline (10mM sodium phosphate buffer of pH 7.2 containing 15 mM NaCl), 10 µl of sample (SDS samples of different concentration) and 250 µl of Stain-All working solution. Read the absorbance at 453 nm making auto zero for blank. Plot a linear regression curve and calculate the concentration of a protein/nucleic acid sample containing the unknown concentration of SDS.
Reference: Analytical Biochemistry (A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration)
Article doi: 10.1016/j.ab.2015.06.013