Methods for Absorbance Assay

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Considerations for use

See considerations listed under the absorbance assay at 280 nm. This method is just as convenient as for absorbance at 280 nm. It may be preferred if there is excessive contamination by nucleic acids, since nucleic acids absorb very little radiation at 205 nm. Setting the wavelength is a bit tricky since 205 nm is right on the shoulder of the protein peak.

Principle

See the discussion for the 280 nm absorbance assay.

Equipment

In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.

Procedure

Include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces. This is necessary for the 205 nm assay because losses are proportionately higher in dilute solutions.
  1. Warm up the UV lamp (about 15 min.)
  2. Adjust wavelength to 205 nm
  3. Calibrate to zero absorbance with buffer solution only
  4. Measure absorbance of the protein solution

Analysis

Protein concentration (mg/ml) = 31(Absorbance at 205 nm).

Comments

Cold solutions can fog up the cuvette, while warm solutions can release bubbles and interfere with the readings. Solutions must be much more dilute than for the A280 assay. Proteins absorb much more strongly at 205 nm, and there is supposedly less variability from protein to protein. In addition to the need for an accurate wavelength setting, stray light can be a major problem. To avoid these problems, use a 10 microgram/microliter solution of bovine serum albumin as a standard. With buffer blank as zero absorbance, determine the concentration of an unknown (concentration between 0 and 10 micrograms/microliter) by interpolation. This is acceptable because of the linear relationship of absorbance and concentration in the 0 to 10 microgram/microliter range.
The problem of an accurate wavelength setting can be avoided by determining absorbance at 210 nm (extinction coefficients range from 20 to 24). However there is less sensitivity and more variation with buffer conditions.

References

  • Scopes, RK. Analytical Biochemistry 59: 277. 1974.
  • Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69. 1990.

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