Spectrophotometry——3

EXERCISE G.2 LOWRY PROTEIN ASSAY

MATERIALS

• 0.15% (w/v) sodium deoxycholate

• 72% (w/v) trichloroacetic acid (TCA)

• Copper tartrate/carbonate (CTC)

• 20% (v/v) Folin-Ciocalteu reagent

• Bovine Serum Albumin (BSA)

• Spectrophotometer and tubes

• Micropipettes

PROCEDURE

1. Prepare standard dilutions of BSA of 25, 50, 75 and 100 µ g/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 µ l of sodium deoxycholate to each tube.

   
   

3. Wait 10 minutes and add 100 µ l of TCA to each tube.

   
   

4. Centrifuge each tube for 15 minutes at 3,000 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 µ l 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.

NOTES

The Lowry method depends on the presence of tyrosine within the protein to be measured. The standard protein must contain approximately the same number of tyrosine residues as the sample, or the procedure will be inaccurate. If there are no tyrosine residues in the sample to be measured, the Lowry method of protein determination is useless, and use should be made of the Bradford assay. In general, the Bradford assay is the method of choice for protein determinations.

EXERCISE G.3 BIURET PROTEIN ASSAY

MATERIALS

• Biuret Reagent

• Bovine serum albumin (BSA)

• Spectrophotometer and tubes

PROCEDURE

1. Prepare standard dilutions of BSA containing 1, 2.5, 5.0, 7.5 and 10 mg/ml protein. Prepare serial dilutions of the unknown samples.

   
   

2. Add 1.0 ml of each of the standards, each sample, and 1.0 ml of distilled water to separate tubes. Add 4.0 ml of Biuret reagent to each tube. Mix by vortex.

   
   

3. Incubate all of the tubes at 37 ° C for 20 minutes.

   
   

4. Turn on and adjust a spectrophotometer to read at a wavelength of 540 nm.

   
   

5. Cool the tubes from Step 3, blank the spectrophotometer and read all of the standards and samples at 540 nm.

   
   

6. Plot the absorbance of the standards vs their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

NOTES

The Biuret reaction was one of the first for the determination of protein concentration. It remains as a rapid determination, but is not very accurate. It is useful during protein separation procedures since there are fewer salt interference reactions than with the Bradford or Lowry techniques. The color formed is stable for about 1-2 hours and consequently all spectrophotometer readings must be made as soon as possible after the incubation step.