What is the function of BCA protein quantification kit (with product manual attached)
The BCA (Bicinchonic acid) method is currently a widely used protein concentration determination method. Based on the biuret reaction, which involves the protein reducing Cu2+to Cu+in an alkaline environment, Cu+forms a purple colored complex with BCA reagent, with a high absorbance at 562 nm. The amount of the reaction product is proportional to the protein concentration. Measure its absorbance at 562 nm and compare it with the standard curve to calculate the concentration of the protein to be tested. This method is fast, sensitive, stable, reliable, and has a small coefficient of variation for different types of proteins. The reagent kit provides protein standards with stable concentration for making standard curves.
BCA protein quantification kit product function
Accurate, sensitive, and with a wide linear range: The protein determination range of BCA reagent is 10-2000 μ g/mL.
Fast: Complete the measurement within 45 minutes, which is 4 times faster and more convenient than the classic Lowry method.
Economical and practical: Conducting measurements in microporous plates can greatly save sample and reagent usage.
Stable effect: not affected by ionic and non-ionic detergents in the sample, the coefficient of variation for detecting different protein molecules is much smaller than that of Coomassie Brilliant Blue.
BCA protein quantification kit usage instructions (detailed instructions can be found in the BCA product manual)
1、 Prepare standard products and working fluids
1. Prepare BSA standard system (linear range 20-2000 μ g/mL or 5-250 μ g/mL standard preparation methods, please refer to
Choose the appropriate method according to habit.
2. Prepare BCA working fluid
(1) Calculate the total volume of BCA working fluid required. Total BCA working fluid volume=(number of standard samples+number of test samples) x number of repetitions x each
BCA working fluid required for the sample.
(2) Preparation of BCA working solution: Add 1 volume of BCA reagent B (A: B=50:1) to 50 volumes of BCA reagent A and mix thoroughly.
2、 Testing method
1. Test tube testing method (sample: BCA working solution=1:20)
(1) Take 100 μ L of standard substance and test sample respectively and add them to the reaction tube.
(2) Add 2.0 mL BCA working solution to each tube and mix well. Select the incubation time and temperature based on the concentration range of the sample to be tested.
Standard incubation method: Incubate at 37 ℃ for 30 minutes or at room temperature for 2 hours (detection range: 20-2000 μ g/mL)
Enhanced incubation method: Incubate at 60 ℃ for 30 minutes (detection range: 5-250 μ g/mL)
(3) Cool to room temperature. Perform detection on a spectrophotometer, set the wavelength to 562 nm, and read all samples within 10 minutes.
(4) Draw a standard curve (X-protein concentration) based on the absorbance of BSA standard (subtracting the OD value of the blank well in the standard to obtain the final reading)
Degree μ g/mL; Y-final OD 562nm). Calculate the protein concentration of the sample based on the standard curve and the dilution factor of the sample.
2. Microplate detection method (sample: BCA working fluid=1:20)
(1) Take 10 μ L of standard and test samples each and add them to the microplate.
(2) Add 200 μ L of BCA working solution to each well and mix thoroughly by blowing with the gun tip. Cover the microplate and incubate at 37 ℃ for 30 minutes.
(3) Cool to room temperature and measure absorbance at a wavelength range of 562 nm on an enzyme-linked immunosorbent assay (ELISA) reader.
(4) Draw a standard curve (X-protein concentration) based on the absorbance of BSA standard (subtracting the OD value of the blank well in the standard to obtain the final reading)
Degree μ g/mL; Y-final OD 562nm). Calculate the protein concentration of the sample based on the standard curve and the dilution factor of the sample.
Precautions for BCA protein quantification kit
1. To ensure the accuracy of protein concentration measurement, it is best to make three replicates and remove outliers if necessary.
2. The BCA method for measuring protein concentration is easily affected by time and temperature, so it is best to make a standard curve for each measurement.
3. When adding samples, it is necessary to add them accurately and avoid the formation of bubbles as much as possible to avoid affecting the measurement (after adding samples, the bubbles can be punctured with a syringe, and the 96 well plate can be placed on a shaker and mixed for a moment to ensure full contact between the sample and the working solution).
4. After incubation, the absorbance should be detected as soon as possible and the detection speed should be accelerated as much as possible to avoid errors.
5. Ensure that all reagents are thoroughly mixed before use and stored at the recommended temperature. The freshness of reagents has a significant impact on experimental results, and expired or improperly stored reagents may lead to inaccurate results.
6. When conducting BCA experiments, strictly follow the order of reagent addition and operation steps to avoid any reversal or omission of steps.
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