Hancock Lab

Protein Assay

Categorized: Protein Purification and Gel Electrophoresis

(Sandermann and Stromiger. 1972. J. Biol. Chem. 247:5123-5131.)

The advantage of this assay over the Lowry assay from which it is derived, is the presence of SDS in reagent A. This allows one to perform protein assays in nearly any detergent (eg., when detergents are used in the purification of outer membrane proteins). In addition, SDS separates membrane proteins from contaminating membrane constituents and denatures the proteins allowing more reproducible results.

Solutions:

2% Na2CO3, 0.02% NaK tatrate, 0.1M NaOH, 1% SDS (added last).
0.5% CuSO4.
On the day of the assay mix 25ml of solution A with 1ml of solution B.
Dilute Folin reagent to 1N with deionized water.

 

Assay:

  1. Wash all glassware with distilled water to remove detergents.
  2. In 13mm borosilicate glass test tubes, add 5 and 10ul of a ten-fold dilution of outer membrane preparation.
  3. Prepare a standard curve by adding 0, 5, 10, 15, 20 and 25ul of 0.1% BSA in water to test tubes as in step 2. Make sure you prepare 2 samples of 0 BSA so you have enough sample for the reference cuvette (1mg/ml).
  4. Add 1ml of solution C, allow to stand for 15 minutes at room temperature.
  5. Add 0.1ml of solution D and vortex the test tube immediately, allow to stand for 30 minutes at room temperature.
  6. Read at 650nm against a reagent blank.

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