An unknown protein was determined using the following method:

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Bradford method

Reagents

 Stock Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G250 in a mixture consisting of 100ml of 85% phosphoric acid, 50ml of 95% ethanol and 50ml 1M NaOH. Store at 4°C until precipitation occurs, at which point it is discarded.

 Working Bradford reagent: Prepare fresh by diluting 10ml of stock Bradford reagent to 250ml with distilled water.

 Stock bovine serum albumin (BSA) solution (10mg/ml): Dissolve 0.2g BSA in 20ml distilled water.

 Working BSA concentration range: 0.5 – 1.25 mg/ml

 

Method

 1.     Prepare the following test tubes:

Table 1: Preparation of test tubes for the Bradford method

 2.    After each addition of the working Bradford reagent, thoroughly mix the resultant solution.

 3.    Incubate the solution at room temperature for 5 min.

 4.    Read the absorbance at 595nm of the blank and test solutions.

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Calculate the protein concentration (in mg/ml) of an unknown sample with an absorbancy value of 0.349 using the standard curve below.

Blank Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Unknown Distilled H20 1.0 ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.9ml 0.25 mg/ml BSA 0.1 ml 0.5 mg/ml BSA 0.1 ml 0.75 mg/ml B SA 0.1 ml 1.0 mg/ml BSA 0.1 ml 1.25 mg/ml B SA 0.1ml Unknown 0.1ml protein Working 4.0ml 4.0ml 4.0ml 4.0ml 4.0ml 4.0ml 4.0ml Bradford