Materials:

1. A UV/Visible spectrophotometer
   
   
2. Pipettes
   
   
3. Pipette tips
   
   
4. Disposable microfuge tubes
   
   
5. Quartz cuvettes (suitable for wavelengths smaller than 205 nm)
   
   
6. Pure protein solution in a buffer (or in water)
   
   
7. The buffer the protein is dissolved in (will act as the blank).

Procedure:

1. Switch ‘ON' the UV/visible spectrophotometer and allow it 30 minutes warm up.
   
   
2. Determine the number of tryptophans, tyrosines, and disulfide linkages present in the protein.
   
   
3. Determine the molar absorption coefficient of the protein at 280 nm using equation 4.4.
   
   
4. Take the buffer used for protein dissolution in the quartz cuvettes.
   1. The volume of buffer has to be sufficient enough to cover the entire aperture the light beam passes through and depends on the capacity of the quartz cuvette; typically cuvettes with 1 ml capacity are used.
      
      
5. Place the cuvettes in the reference cell and sample cell slots in the spectrophotometer.
   
   
6. ‘ZERO' the baseline for the 250 – 350 nm range.
   
   
7. Remove the quartz cuvette placed in the sample cell slot and discard all the contents.
   
   
8. Add the same volume of the given protein solution into the cuvette and place it back in the sample cell slot.
   
   
9. Record the absorbance at 280 nm and 330 nm
   1. Proteins do not absorb at wavelengths higher than 320 nm; any absorbance obtained at 330 nm therefore arises due to scattering.
      
      
   2. If the absorbance at 280 nm does not lie between 0.05 – 1.0, dilute the protein solution in the same buffer so as to obtain an absorbance in this range.
      
10. Switch off the spectrophotometer.
    
    
11. Take out the quartz cell and clean them using detergent solution and deionized water.