BCA Protein Assay Kit

Cat. No.: B665595

Overview

The BCA (Bicinonic acid) method is a widely used method for measuring egg protein concentration. Based on the biuret reaction, which involves the reduction of Cu2+to Cu+by egg white in an alkaline environment, a purple blue complex is produced, with a high absorbance value at 562nm. The amount of the reaction product is positively correlated with the concentration of egg white. The BCA protein concentration determination method has achieved the simplicity, sensitivity, speed, and stability of protein concentration determination. The egg white standard provided in the reagent kit provides convenience for users to create standard curves. The BCA egg protein concentration determination kit can be used for nucleic acid detection or for microplate detection. The former requires a relatively large amount (100 μ L) of egg samples, but due to its use in detection, the ratio of egg samples to BCA working solution is 1:20 (v/v), thereby reducing the impact of interfering substances. The latter is easy to operate and only requires a small amount (10-25 μ L) of egg yolk sample. However, due to its 1:8 (v/v) ratio between the egg white sample and BCA working solution during detection, it to some extent limits the tolerance concentration of interfering substances and reduces the minimum detection level. 

Component 

Composition	Specifications 500T	Storage
BCA reagent A	100mL	4℃
BCA reagent B	3mL	4℃
Protein standards（BSA）	2×1mL（5mg/mL）	-20℃
PBS  Solution	10mL	4℃

Product features
1) High sensitivity, with a minimum detection protein content of 0.2 μ g and a detection concentration lower limit of 10 μ g/mL (with a good linear relationship in the concentration range of 20~1000 μ g/mL).
2) Fast speed and shorter color development time than typical BCA protein concentration determination kits. The detection speed is about 4 times faster than the traditional Lowry method.
3) The linear range is wide, with a good linear range within the concentration range of 20-1000 μ g/mL.
4) The biggest advantage of using BCA method to determine protein concentration is that it is not affected by most chemical substances in the sample. The determination of protein concentration can tolerate high concentrations of descaling agents and is compatible with up to 5% SDS in the sample; 5% Triton X -100; 5% Tween 20, 60, 80. However, due to the influence of chelating agents and slightly higher concentrations of reducing agents, it is necessary to ensure that EDTA is below 10mM, there is no EGTA, and dithiothreitol is below 1mM; β - mercaptoethanol is less than 0.01%. Please refer to Appendix 1 of 7 for details.
5) Comparison between Bradford method and BCA method

Method	Bradford method	BCA method
Sensitivity	1~5 μg	0.5~20 μg
Time	5~15 min	40~60 min
Wavelength	595 nm	562 nm
Detecting proteins of different categories	High coefficient of variation	Low coefficient of variation
Absorbing stability	Stable within 1 hour	Over time

Operating instructions
BCA working solution preparation:
Mix reagent A and reagent B in a volume ratio of 50:1 to prepare BCA working solution. Mix 50mL of reagent A with 1mL of reagent B to prepare 51mL of BCA working solution. When the two are mixed, precipitation will form, and after thorough mixing, the precipitation will disappear.
Microporous plate measurement program: (working range 20-2000 μ g/ml)
1. Preparation of protein standard: Dissolve the protein standard completely at room temperature, take 20 µ l 5mg/ml BSA protein standard solution and dilute it with PBS solution to 100 µ l to achieve a final concentration of 1.0 mg/ml.
2. Prepare BSA standard measurement solution according to the following table:

Number	0	1	2	3	4	5	6	7	8
/	1 mg/ml BSA  Standard solution  μL	1 mg/ml BSA  Standard solution  μL	1 mg/ml BSA  Standard solution  μL	1 mg/ml BSA  Standard solution  μL	1 mg/ml BSA  Standard solution  μL	1 mg/ml BSA  Standard solution  μL	1 mg/ml BSA  Standard solution  μL	5 mg/ml BSA  Standard solution  μL	5 mg/ml BSA  Standard solution  μL
BSA Standard solution  µl	0	0.5	2.5	5	10	15	20	6	8
PBS Solution  μl	20	19.5	17.5	15	10	5	0	14	12
BSA Final concentration  μg/ml	0	25	125	250	500	750	1000	1500	2000
Total volume  µl	20 µl	20 µl	20 µl	20 µl	20 µl	20 µl	20 µl	20 µl	20 µl

3. Add an appropriate volume of the test sample to the microplate and supplement it with PBS to 20 µ l
4. Add 200 μ L of BCA working solution to the microplate and mix well, then let it stand at 37 ℃ for 30 minutes; Note: It can also be left at room temperature for 2 hours or at 60 ℃ for 30 minutes. When measuring protein concentration using the BCA method, the color will deepen over time, and the color reaction will accelerate with increasing temperature. If the concentration is low, it is suitable to incubate at a higher temperature or extend the incubation time appropriately.
5. Measure the absorbance value at 562 nm and record the reading; Use the light absorption value of the sample without BSA as the blank control.
6. Draw a standard curve with A562 as the vertical axis and BSA content as the horizontal axis, and calculate the protein concentration in the sample If the protein concentration is not within the standard curve range, please dilute the sample and retest.
Test tube testing procedure: (working range 20-1000 μ g/ml)
1. Preparation of protein standards:
Dissolve the protein standard completely at room temperature, take 150 µ L 5mg/ml BSA protein standard solution, add 600 μ L LPBS solution and dilute to 750 µ l to achieve a final concentration of 1.0 mg/ml.
2. Prepare BSA standard measurement solution according to the following table:

Number	0	1	2	3	4	5	6	7	8
/	1 mg/ml BSA Standard solution  μl	1 mg/ml BSA Standard solution  μl	1 mg/ml BSA Standard solution  μl	1 mg/ml BSA Standard solution  μl	1 mg/ml BSA Standard solution  μl	1 mg/ml BSA Standard solution  μl	1 mg/ml BSA Standard solution  μl	5 mg/ml BSA Standard solution  μl	5 mg/ml BSA Standard solution  μl
BSA Standard solution  µl	0	2.5	12.5	25	50	75	100	30	40
PBS  Solution μl	100	97.5	87.5	75	50	25	0	70	60
BSA Final concentration  μg/ml	0	25	125	250	500	750	1000	1500	2000
Total volume  µl	100 µl	100 µl	100 µl	100 µl	100 µl	100 µl	100 µl	100 µl	100 µl

3. Add an appropriate volume of the sample to be tested into the test tube and supplement it with PBS to 100 µ l;
4. Add 2ml of BCA working solution to the test tube, mix well, and let it stand at 37 ℃ for 30 minutes;
5. 6 steps as above

Matters needing attention

1) When using the BCA egg size determination method, the color will deepen over time, and the speed of the color reaction is related to temperature. It is important to maintain timing and temperature to ensure accurate quantification.
2) If BCA reagent A or reagent B is found to have precipitates, it should be stored for a period of time. Please incubate at 37 º C and stir to fully dissolve. If bacterial contamination is found, it should be discarded.
3) Standardize experimental operations to improve the accuracy of sample loading.
4) Each measurement requires a corresponding standard curve, as the visible reaction is related to changes in temperature and time. Accurate egg quantification should be done with a standard curve every time.
5) All products of our company are limited to research and development purposes only, and must be operated by professional personnel while wearing masks/gloves/lab clothes and complying with the safety operating procedures of the biological laboratory!

Storage and Shipping

Storage

Store at 2-8°C

Shipped In

Wet ice

References

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