An unknown protein was determined using the following method: Bradford method Reagents Stock Bradford reagent: Dissolve 100
Question:
An unknown protein was determined using the following method:
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.
Calculate the protein concentration (in mg/ml) of an unknown sample with an absorbancy value of 0.349 using the standard curve below.
Introduction to Chemical Engineering Thermodynamics
ISBN: 978-0071247085
7th edition
Authors: J. M. Smith, H. C. Van Ness, M. M. Abbott