BCA Protein Quantitative Detection Kit (Ready to use)

The BCA protein quantification kit is a kit based on BCA ( Bicin-choninic Acid ), which uses colorimetry to determine the total protein concentration. It is one of the widely used protein quantification methods. The principle is that under alkaline conditions, the peptide bond in the protein reduces Cu2 + to Cu +, and BCA chelates Cu + as a chromogenic agent to produce a blue-purple complex with an absorption peak at 562 nm. The depth of the color is proportional to the protein concentration, and the protein content can be determined according to the absorption value. The BCA protein quantitative kit ( i.e.type ) is a protein standard solution ( BSA solution ) containing a series of concent Components Components B736762-500T A.Reagent A 100 mL B.Reagent B 1.5 mLx2 C.BSA standard①(2000 μg/mL) 1 mL D.BSA standard②(1500 μg/mL) 1 mL E.BSA standard③(1000 μg/mL) 1 mL F.BSA standard④(750 μg/mL) 1 mL G.BSA standard⑤(500 μg/mL) 1 mL H.BSA standard⑥(250 μg/mL) 1 mL I.BSA standard⑦(125 μg/mL) 1 mL J.BSA standard⑧(0 μg/mL) 1 mL Points for attention1.Reagent A can be completely dissolved at 37 °C if flocculent crystallization occurs at low temperature, which does not affect its use. 2.The protein standard solution ( BSA ) should be put in advance to room temperature to melt, and mixed upside down ; 3.It is recommended that the standard curve should be drawn every time the protein sample is measured to make the measurement result accurate ; 4.In order to improve the accuracy of the sample measurement results, the sample concentration should not be too high or too low, otherwise it needs to be diluted or concentrated in advance ;5.For your safety and health, please wear experimental clothes and disposable gloves. Instruction 1 Preparation of protein standard solution (BSA)Take the matching and ready to use BSA standard product out of the -20 ℃ refrigerator and melt it at room temperature. Invert it upside down and mix well for later use.II Preparation of BCA working solution1. Calculate and determine the total volume of working fluid required:The total amount of BCA working solution=(number of BSA standard samples+number of test samples) x number of wells x volume of BCA working solution required for each sample. For example, for this reagent kit quality inspection, the number of BSA standard samples required is 8, the number of test samples is 3, and the number of wells is 3.The total amount of BCA working solution=(8+3) x 3 x 200 μ L (volume of each sample working solution)=6.6 mL2. Based on the calculated volume of BCA working solution, prepare BCA working solution with reagent A and reagent B in a 50:1 volume ratio, and mix thoroughly. Note: 1) Due to possible errors in sample addition, it is recommended to prepare 1-2 more holes when preparing the working solution;2) When reagent B is added to reagent A, turbidity can be observed initially, which is a normal phenomenon. After stirring, the turbidity will quickly disappear and an apple green clear working solution is obtained.III Protein concentration measurement (96 well plate as an example)1. Take 20 μ L of BSA standard at each dilution concentration and add it to a 96 well transparent plate;2. Add 200 μ L of working solution to each well and mix well. Cover with a 96 well plate and incubate at 37 ℃ for 30 minutes;3. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of A562 or other wavelengths between 540 and 590 nm for each sample and BSA standard;4. Draw a standard curve and calculate the protein concentration in the sample.Note: 1) The absorbance of the blank control needs to be subtracted from the A562 of each sample and BSA standard;2) When processing data, it is necessary to remove obvious outliers or numerical values;3) If the sample concentration is too high and exceeds the upper limit (2000 μ g/mL), it should be diluted appropriately. If the sample concentration is too low, it is recommended to concentrate it appropriately.Appendix: Tolerance concentration of interfering substances Interference substances Tolerance concentration Interference substances Tolerance concentration Ammonium sulfate 1.5 M Deoxycholic acid 5% EPPS ，pH 8.0 100 mM NP-40 5% Glycine·HCI,pH2.8 100 mM SDS 1% Guanidine·HCI 4 M Triton X-100 5% HEPES ，pH 7.5 100 mM Tween-20 5% Imidazole ，pH 7.0 50 mM EDTA 2 mM MOPS ，pH7.2 100 mM DTT 0. 1 mM PIPES ，pH6.8 100 mM Glucose 10 mM Sodium azide 0.20% 2-Mercaptoethanol 0.01% Sodium bicarbonate 100 mM DMSO 10% Sodium chloride 1 M Ethanol 10% Tris 250 mM Glycerol 10% EGTA 1 mM β - mercaptoethanol 150 μM.