BCA Protein Assay Kit

Product contentComponent500 microplate assaysor 50 tube assaysBCA-A2×50 mLBCA-B3 mLBSA Standard Solution（2mg/mL）2 mProduct IntroductionThe BCA protein quantification method is currently one of the widely used protein quantification methods. This product is developed based on the BCA (Bicin chonic Acid) method, which enables rapid, stable, and sensitive concentration determination of proteins. The principle is that the peptide chain structure in protein molecules can form complexes with Cu2+in alkaline environments, while reducing Cu2+to Cu+. BCA reagents can sensitively and specifically bind with Cu+to form stable colored complexes. There is a high light absorption value at 562 nm, and the depth of color is proportional to the protein concentration. The protein content can be determined based on the magnitude of the absorption value. This reagent kit contains bovine serum albumin (BSA) solution as a protein standard solution, with a measurement range of 20-2000 μ G/mL.matters needing attention1. This product can be measured for protein concentration using a spectrophotometer (test tube detection method) or an enzyme-linked immunosorbent assay (microplate detection method).2. It is recommended to draw a standard curve for each protein sample measurement to obtain accurate data.3. The diluent of BSA standard should be consistent with the diluent of the test sample (can be diluted with 1 × PBS or 0.9% physiological saline).4. If the sample to be tested contains a significant amount of interfering substances, the Bradford protein quantification kit or other protein quantification products can be used.Operation steps1. Dilute BSA standard: Dilute BSA standard with the same diluent as the protein sample to be tested as shown in the table below. Pipe No. Dilution dosage（μL） BSA Standard Dosage（μL ） BSA standard final concentration（μg/μL ） A 0 100 2 B 200 200 1 C 200 200（Take from tube B） 0.5 D 200 200（Take from tube C） 0.25 E 200 200（Take from tube D） 0.125 F 200 200（Take from tube E） 0.0625 G 200 0 0Total amount of BCA working solution=(number of BSA standard samples+number of unknown samples) x number of wells x volume of BCA working solution per sampleFor example, the number of BSA standard samples is 7, the number of unknown samples is 2, and the number of re wells is 3.Test tube testing method:Total amount of BCA working solution=(7 BSA standard samples+2 unknown samples) x 3 wells x 2 mL/volume of working solution per sample=54 mLMicro pore detection method:Total amount of BCA working solution=(7 BSA standard samples+2 unknown samples) x 3 wells x 200 μ L/Volume of each sample working solution=5.4mL 2) Based on the calculated total amount of BCA working solution required, prepare BCA working solution and BCA-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 BCA working solution.2) The newly prepared BCA working solution can be stably stored for 24 hours under room temperature sealing conditions.3. Quantitative testing3a Test tube detection method (protein concentration detection range: 20-2000) μ G/mL1) According to the above table, dilute the A-G BSA standard and the protein sample to be tested (stock solution or dilution solution) by 100 each μ LAdd them separately to the labeled test tubes. Recommend 2-3 parallel reactions for each measured sample.2) Add 2mL of BCA working solution to each test tube, mix well, and incubate in a 37 ℃ water bath for 30 minutes. Cool each tube to room temperature and complete the test within 3-5 minutes.3) Measure the absorbance values of each sample and BSA standard at 562 nm using a spectrophotometer, and keep records.4) Draw a standard curve and calculate the protein concentration in the sample.3b Micro pore detection method (protein concentration detection range: 20-2000) μ G/mL1) According to the above table, dilute the A-G BSA standard and the protein sample to be tested (stock solution or dilution solution) by 25 each μ Add l separately to the marked 96 well plate micropores. Recommend 2-3 parallel reactions for each measured sample. 2) Add 200 to each hole μ Mix BCA working solution thoroughly, cover with a 96 well plate cover, incubate at 37 ℃ for 30 minutes, cool to room temperature, and complete the test within 3-5 minutes.3) Measure the absorbance values of each sample and BSA standard in the range of 540-590 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and keep records.4) Draw a standard curve and calculate the protein concentration in the sample.Note: 1) When using the BCA method to determine protein concentration, the absorbance value will continuously deepen over time. Therefore, all sample measurements need to be completed within 3-5 minutes, otherwise it will affect the accuracy of protein quantification.2) It is recommended to draw a standard curve based on the absorbance reading after removing background values.3) If the standard reading deviates significantly from the linear curve due to operational errors, it should be discarded.4) The unknown sample concentration can be calculated from the standard curve equation, and the actual concentration needs to be multiplied by the dilution factor of the sample.5) If the obtained protein concentration is not within the detection range, please dilute the sample again and retest.4. Examples of quantitative analysis results for BCA protein:Results of microplate detection method protein concentration（μg/μL） Original absorbance value Absorbance value after removing background value 0 0.086(Background value) 0 0.125 0.123 0.037 0.25 0.28 0.194 0.5 0.551 0.465 1 1.068 0.982 2 2 1.913.