Application

This kit can be used to measure the total protein in serum, plasma and animal tissue samples. 

Detection principle

Coomassie brilliant blue G-250 is red under the free state, and it has the maximum absorbance at 465 nm. When the Coomassie brilliant blue G-250 combined to protein, the compound will have the maximum at 595 nm. The absorbance value is directly proportional to the protein content, so the concentration of total protein can be calculated directly by measuring the OD value at 595 nm.

Experimental instrument

Micropipette, Vortex mixer, Centrifuge, Spectrophotometer (595 nm)

Sample preparation

1. Serum (plasma) samples: detect directly. If the concentration of sample is beyond the linear range, please dilute the sample with PBS (0.01 M, pH 7.4) and then carry the assay.

2. Tissue samples: Weigh 0.02-1 g of tissue accurately. Add 9 times the volume of PBS (0.01 M, pH 7.4) according to the proportion of Weight (g): Volume (mL) =1:9. Mechanically homogenize the sample in ice water bath. Centrifuge at 1500 g for 10 min. Take the supernatant and preserve it on ice for detection.

Operation steps

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

1. Blank tube: add 3000 μL of reagent 1 application solution and 50 μL of double distilled water into a 5 mL EP tube and mix fully.

Standard tube: add 3000 μL of reagent 1 application solution and 50 μL of Standard solution into a 5 mL EP tube and mix fully.

Sample tube: add 3000 μL of reagent 1 application solution and 50 μL of sample into a 5 mL EP tube and mix fully.  

2. Stand the tubes at room temperature for 10 min. Set the spectrophotometer to zero with double-distilled water and measure the OD value of each tube at 595 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 26 μg/mL.

2. The intra-assay CV is 1.8% and the inter-assay CV is 2.3%.

3. The recovery of the kit is 97%.

4. The linear range of the kit is 26-1200 μg/mL.

Notes

1. The kit is for scientific research only.

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

3. The valid of kit is 6 months.

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