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  Section: Biotechnology Methods » Biochemistry
 
 
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Estimation of Protein by the Lowry Protein Assay

 
     
 

Materials

  1. 0.15% (w/v) sodium deoxycholate
  2. 72% (w/v) Trichloroacetic acid (TCA)
  3. Copper tartrate/carbonate (CTC)
  4. 20% (v/v) Folin-Ciocalteu reagent
  5. Bovine serum albumin (BSA)
  6. Spectrophotometer and tubes
  7. Micropipettes

Procedure

  1. Prepare standard dilutions of BSA of 25, 50, 75, and 100 mg/mL. Prepare appropriate serial dilutions of the sample to be measured.
  2. Place 1.0 mL of each of the above into separate tubes. Add 100 mL of sodium deoxycholate to each tube.
  3. Wait 10 minutes and add 100 mL of TCA to each tube.
  4. Centrifuge each tube for 15 minutes at 3000 xg and discard the supernatant.
  5. Add 1.0 mL of water to each tube to dissolve the pellet. Add 1.0 mL of water to a new tube to be used as a blank.
  6. Add 1.0 mL of CTC to each tube (including the blank), vortex and allow to set for 10 minutes.
  7. Add 500 mL Folin-Ciocalteu to each tube, vortex and allow to set for 30 minutes.
  8. Turn on and zero a spectrophotometer to a wavelength of 750 nm. Use the blank from step 7 to adjust for 100% T.
  9. Read each of the standards and samples at 750 nm.
  10. Plot the absorbance of the standards vs their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.
 
     
 
 
     




     
 
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