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  Section: Biotechnology Methods » Biochemistry
 
 
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Protein Assay by Bradford Method

 
     
 

Materials

  • Lyophilized bovine plasma gamma globulin or bovine serum albumin (BSA)
  • Coomassie Brilliant Blue
  • 0.15 M NaCl
  • Spectrophotometer and tubes
  • Micropipettes

Procedure (Standard Assay, 20–150 µg protein; 200–1500 µg/mL)

  1. Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750, and 1500 µg BSA/µL. Also prepare serial dilutions of the unknown sample to be measured.
  2. Add 100 mL of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20).
  3. Add 5.0 mL of Coomassie Blue to each tube and mix by vortex, or inversion.
  4. Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube from step 3, which contains 0 BSA.
  5. Wait 5 minutes and read each of the standards and each of the samples at a 595-nm wavelength.
  6. Plot the absorbance of the standards versus their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

Procedure (Micro Assay, 1–10 µg protein)

  1. Prepare standard concentrations of BSA of 1, 5, 7.5, and 10 µg/mL. Prepare a blank of NaCl only. Prepare a series of sample dilutions.
  2. Add 100 µL of each of the above to separate tubes (use microcentrifuge tubes). Add 1.0 mL of Coomassie Blue to each tube.
  3. Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5-mL cuvettes.
  4. Wait 2 minutes and read the absorbance of each standard and sample at 595 nm.
  5. Plot the absorbance of the standards versus their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
 
     
 
 
     




     
 
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