The Lowry method is a standardized, rapid method for protein quantification and has been widely used to remove interfering substances by protein precipitation prior to testing. Source: Handbook of Protein Technology

Operation method

Lowry assay

Principle

The Lowry method is also known as the Folin-phenol reagent method. The copper-protein complex is first formed in alkaline solution, and then this complex reduces phosphomolybdic acid-phosphotungstic acid reagent (Folin-phenol upper level), which produces a dark blue color of molybdenum blue and tungsten blue complex, and this dark blue complex has the maximum absorption peak at 745-750 nm, and the depth of the color (absorption value) is directly proportional to the concentration of the protein, so that it can be calculated according to the magnitude of the light absorption value at 750 nm. The color (absorption value) is directly proportional to the protein concentration.

Materials and Instruments

Protein samples to be measured
Copper sulfate pentahydrate Sodium citrate Sodium carbonate NaOH Folin-phenol reagent
Spectrophotometer Colorimetric cup Pipettes Guns and spiking guns Test tubes (3 to 5 ml) Test tube rack Oscillator

Move

1. Solution

1.1 Solution A, 100 ml

0.5 g _CUSO4-5H2O

1 g _Na3C6H5O7 ( _-2H2O )

Add double-distilled water to 100 ml, the solution can be stored at room temperature for a long time.

1.2 Solution B, 1 L

20 g _Na2CO3

4 g NaOH

Add distilled water to 1 L. The solution can be stored at room temperature for a long time.

1.3 Solution C, 51 ml

Folin solution A

50 ml of solution B

1.4 Solution D, 20 ml

10 ml Folin-Phenol Reagent

10 ml double-distilled water

2. Detection

2.1 Dilute sample to 0.5 ml with double-distilled water;

2.2 Add 2.5 ml of Solution C. 2.3 Mix well;

2.2 Add 2.5 ml of Solution C. 2.3 Mix well and let stand at room temperature for 5-10 min;

2.4 Add 0.25 ml of solution D and mix well;

2.5 After 20-30 min, measure the _A750 value on a spectrophotometer. 3. Purification from interfering substances

3. Additional steps for purification of protein samples from interfering substances

Deoxycholic acid-trichloroacetic acid (DOC-TCA) precipitation. This method can be used when the concentration of the solution in which the protein is measured is less than 1 ug/ml.

Reagents required: 0.15% (w/V) DOC: 72% TCA

3.1 Add 0.1 ml of 0.15% DOC to 1 ml of protein sample;

3.2 Shake and leave at room temperature for 10 min;

3.3 Add 0.1 ml of 72% TCA, shake well, and centrifuge for 5-30 min at 1000-3000 × g. Longer centrifugation times are required if a fixed-angle rotor head is used, and for low temperatures or large volumes;

3.4 Tilt and discard the supernatant;

3.5 Dissolve the precipitate directly in Solution C.

Caveat

1. the color saturates 20-30 min after the reagent is added, and the color signal decreases by 1% every hour thereafter. 2. many interfering substances reduce the color response, while descaling agents cause a slight increase in color.

2. many interfering substances reduce the color response, while descaling agents cause a slight increase in color. 3. high concentrations of salt may cause precipitation.

3. high concentration of salt can cause precipitation.

4. chloroform extraction removes lipids from the solution, and the color signal decreases by 1% every hour thereafter.+Chloroform extraction removes lipids from the solution, and centrifugation removes turbidity in the presence of K+ or Triton X-100.

5. interference from detergents, sucrose and EDTA can be eliminated by adding SDS to Lowry's reagent.

6. the extinction coefficient of protein-dye complexes is normally ≤ 1.

2.

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