Application

The kit can be used to measure total protein (TP) content in serum (plasma), tissue and cultured cells. 

Detection principle

Any compound that contains two -CONH2 in the molecule can react with alkaline copper solution to form a purple complex, which is known as the biuret reaction. Many peptide bonds (-CONH-) in protein molecules can perform this reaction, and the color degree of all kinds of proteins are essentially the same.

Experimental instruments

Test tube, Micropipettor, Vortex mixer, 37℃water bath, Spectrophotometer (540 nm)

Preparation of sample

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment:

1.    Serum: detect directly.

2.    10% tissue homogenate: Accurately weigh the tissue, add 9 times the volume of 1×PBS (0.01 M, pH 7.4) according to the ratio of Weight (g): Volume (mL) =1:9. Take the mechanical homogenization in ice water bath. Centrifuge at 10000 g for 10 min, then take 50 μL of the supernatant for detection.

Operation procedures

1.     Blank tube: Add 50 μL of PBS into a 5 mL EP tube.

Standard tube: Add 50 μL of 50 g/L Protein standard into a 5 mL EP tube.

Sample tube: Add 50 μL of Sample into a 5 mL EP tube.

2.     Add 2.5 mL of biuret working solution into each tube, mix fully with a vortex mixer.

3.     Incubate the tubes at 37℃ for 30 min, then cool the tubes with running water.

4.     Set the spectrophotometer to zero with double-distilled water and measure the absorbance at 540 nm with 1 cm diameter cuvette.

Note: It can be refer to the following operating table

Technical parameters

1.  The sensitivity of the kit is 0.373 g/L.

2.  The intra-assay CV is 1.2 % and the inter-assay CV is 2.6%.

3.  The recovery of the kit is 99 %.

4.  The detection range of the kit is 0.373-80 g/L.

Note

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of the kit is 6 months.

4. Do not use components from different batches of kit.

5. If the protein is higher than 80 g/L, please dilute the sample with 1×PBS (0.01 M,pH 7.4) and re-test. If the protein content is lower than 5 g/L, it is suggested to detect the sample with BCA method (E-BC-K318) or Coomassie brilliant blue method (E-BC-K168).