Scopes published a simple method for determining the extinction coefficient (E) of proteins (Scopes, 1974). This method is based on the fact that although most of the absorption of proteins at 205 nm is due to peptide bonds, some of the absorption depends on the amino acids in them. This experiment was obtained from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.

Operation method

Determination of extinction coefficient (Scopes method) Experiments

Materials and Instruments

Dialysis buffer Bovine serum albumin (BSA) or σ32 solution
Clean quartz cuvette Spectrophotometer

Move

Materials and equipment

Bovine serum albumin (BSA) or ^σ32 solution at a concentration of approximately 1mg/ml

Clean quartz cuvette

Spectrophotometer,

reagent readable at 205nm and 280nm

Dialysis buffer

(For formulations, see "Preparation of Reagents," PP.184-189)

Operating Procedures

1) Dialyze 2X400 ml of dialysis buffer with BSA solution (~1mg/ml) or unknown concentration of pure protein solution for 3~5 h with sufficient agitation.

2) Determine _A280nm (should be in the range of 0.25~1.0).

3) Carefully dilute the protein solution 30 times with dialysis buffer (e.g., 200ul of sample with 5.8 ml of dialysis buffer) and measure _A205nm.

4) Calculate E(1 mg/ml _)205nm using the formula E(1 mg/ml _)205nm=27.0+120( _A280/A205 ).

5) Calculate: protein concentration = _A205/E (1 mg/ml _)205nm=

6) Calculate: E(1 mg/1 ml _{)280nm=A280/protein} concentration=

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