Experiment on the determination of protein content by colorimetric method of micropanel of Caumas Brilliant Blue
Experiment on the determination of protein content by colorimetric method of micropanel of Caumas Brilliant Blue
This experiment is mainly used to determine the protein concentration.
Operation method
Experiment on the determination of protein content by colorimetric method of micropanel of Caumas Brilliant Blue
Principle
Khomas Brilliant Blue G-250 exists in two different color forms, red and blue. It binds to proteins through van der Waals forces, and within a certain range of protein concentration, the binding of protein and dye conforms to Beer?s law. After the dye binds to the protein, the color changes from red to blue, and the maximum light absorption changes from 465nm to 595nm. The amount of protein bound to the dye can be determined by measuring the increase in light absorption at 595nm. The binding of protein and dye is a very fast process, the reaction can be complete in about 2 min, presenting the maximum light absorption, and can be stabilized for 1h, after which the protein and dye complexes polymerize and precipitate out. The protein-dye complex has a high extinction coefficient, which makes it highly sensitive in the determination of protein concentration. 0.275 light absorption value change was observed in the determination of 5 μg/mL of protein in the solution, which is 4 times more sensitive than Lowry's method, and the range of the determination was 10-100 μg of protein, and the range of the microdosimetry method was 1-10 μg of protein. This reaction is reproducible, accurate and linear. This method has few interferences, and studies have shown that NaCl, KCl, MgCl2, ethanol, and (NH4)2SO4 do not interfere. Strong alkaline buffer in the determination of some color interference, which can be used to appropriate buffer control deduction of its effect. Because of the simplicity of the method, a single color developer, rapid reaction, etc., the CBB method is more commonly used. However, for the determination of large quantities of samples with small amounts of protein, the macronutrient method (MC) is time-consuming and reagent-intensive, and the protein conjugate with CBB is easily adsorbed on the colorimetric cup, which is not easy to clean, and the results will be affected by the reuse of the method. Therefore, we established the microplate colorimetric method (MPC) for the determination of CBB on the enzyme-linked immunoassay (ELISA) instrument.
Materials and Instruments
Mouse Move 1. Sample preparation Caveat 1. Tris, acetic acid, 2-mercaptoethanol, sucrose, glycerol, EDTA and trace amounts of detergents such as Triton X-100, SDS, and glass detergents have a small amount of color interference, which can be easily removed by using appropriate buffer controls. However, the presence of a large number of decontaminants on the color is too great an influence and is not easy to eliminate. 2. If the measurement requirements are very strict, the light absorption can be measured within 5-20min after the reagents are added, because the color is the most stable during this time. 3. the protein-dye complex will be adsorbed on the wall of the colorimetric cup, which will have some influence on the result, so the blue colorimetric cup should be washed with ethanol after each sample of the constant method. 4. If a full-wavelength ELISA is available, the measurement wavelength should be 600 nm. Common Problems Source Experimental Basic Medicine. For more product details, please visit Aladdin Scientific website.
Standard Bovine Serum Albumin (BSA) Solution Kaumas Brilliant Blue Reagent Physiological Saline
Test tubes Test tube racks Pipettes Pipettes Glass homogenizer Ophthalmic scissors Tweezers Balance Spectrophotometer 96-well plate Enzyme immunoassay tester
Mice were killed by cervical dislocation, and the liver tissue was dissected out, and 5g/L of homogenate was prepared in saline. If there is any lumpy insoluble material, it will cause spiking error, and should be centrifuged at 500 rpm for 5 min, and the supernatant should be taken for spare.
2. Plotting of standard curve
(1) Constant method.
Add BSA standard solution into test tubes: 12.5, 25, 50, 75, 100, 125, 150 μl (i.e., containing 6.25, 12.5, 25, 37.5, 50, 62.5, 75 μg of BSA respectively), then add saline to make the volume 200 μl, and add 200 μL of saline to the blank tube, then add 4000 μl of Caulmers Blue color solution, shake well, and measure the optical density at 595 nm. After mixing, each tube was filled with Caulmers Brilliant Blue 4000 μl, shaken well, and left at room temperature for 5 min, the optical density was measured at 595 nm.
(2) Trace method.
Add 1.25, 2.50, 3.75, 5.00, 6.25, 7.50 μL of BSA standard solution (containing 0.625, 1.250, 1.875, 2.500, 3.125, 3.750 μg of BSA) into the 96-well plate, and then replenish with saline to make the volume of 10 μl of liquid in each well, and add 10 μl of saline to blank wells only. After shaking slowly, 200 μl of CBB color solution was added to each well, and the absorbance was measured directly at 600 nm on an enzyme immunoassay instrument after 5 m in at room temperature. The results were analyzed by linear regression and plotted.
3. Determination of protein content of samples
The same method as above, take the appropriate volume of the unknown sample solution, so that the measured value is within the linear range of the standard curve. The protein concentration (mg/mL) of the unknown sample was calculated from the regression equation based on the A595nm and 600 nm values determined respectively.
4. The protein concentration of the sample was calculated from the measurement results and the correlation between the two measurement methods was shown statistically.