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Star Republic: Guide for Biologists

Estimation of protein concentration

The amino acids tyrosine and tryptophan absorb light at 280 nm. Unless the molar absorptivity or extinction coefficient of a protein is known, UV absorbance measurement can only yield an estimate of protein concentration.

1. Turn on OD absorbance spectrometer. Turn on UV source. Wait 5-10 minutes for the UV source to stabilize.

2. Rinse the quartz measurement cuvette with dH2O. Note plastic cuvettes are not UV transparent, thus can't be used for UV measurement.

3. Adjust UV wavelength to 280 nm, fill the quartz cuvette with water. Zero the spectrometer, i.e., the OD reading for water should be 0.

4. Measure UV absorbance at 280 nm for the protein sample.

5. To correct for nucleic acid contaimnations, repeat steps 2-4 at the wavelength 260.

6. Estimate protein concentration using the OD calculator of BioToolKit or the following formula (for cuvette with 1-cm light path):

Protein concentration ( mg/ml ) = 1552 x A280 - 757.3 x A260

7. Clean up the cuvette, turn off the UV source and the spectrometer.