Practical Protein Chemistry - A. Darbre 1989
Analytical Methods
Quantitative Protein Determination
Atomic Absorption Spectrophotometry Method
The method allows the determination of Proteins in colored or opalescent solutions [92].
Reagent. Add 10 ml of 0.20 M CuSO4 to 590 ml of 0.167 M NaOH containing 2 g of Na2K tartrate tetrahydrate. AG2-X8 ion-exchange resin.
Class="center">Table 8.3. Determination of protein amount by comparing optical densities at 280 and 260 nm
|
A280/260 |
Nucleic acid, % |
F |
|
1,75 |
0,0 |
1,116 |
|
1,52 |
0,5 |
1,054 |
|
1,36 |
1,0 |
0,994 |
|
1,16 |
2,0 |
0,899 |
|
1,03 |
3,0 |
0,814 |
|
0,939 |
4,0 |
0,743 |
|
0,874 |
5,0 |
0,682 |
|
0,822 |
6,0 |
0,632 |
|
0,784 |
7,0 |
0,585 |
|
0,753 |
8,0 |
0,545 |
|
0,730 |
9,0 |
0,508 |
|
0,705 |
10,0 |
0,478 |
|
0,645 |
14,0 |
0,377 |
|
0,595 |
20,0 |
0,278 |
Varian-Techtron atomic absorption spectrophotometer, model 1000 with an air-acetylene flame.
Procedure. Add 1 ml of Cu reagent to 1 ml of protein solution (0.05–1.0 mg/ml). Mix. After 2–5 min, add 250 mg of resin and shake intermittently for 5 min. After the resin settles, take 1 ml of the supernatant and add it to 3 ml of distilled Water. Determine the copper content using an atomic absorption spectrophotometer at 324.8 nm. Prepare a reagent blank solution and construct a calibration curve for BSA (0.05–1.0 mg/ml). To reduce instrument noise and protect the instrument from clogging between sample measurements, aspirate 0.5 ml of glacial acetic acid. The presence of chlorophyll, heme, ruthenium, and other Cofactors does not interfere with the determination. The method is used when other Methods are not applicable. The protein solution must contain <0.4 M salts or Amino Acids. Insoluble Proteins can be brought into solution using 1% (w/v) SDS.
Last update: 06/08/2026
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