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BCA protein quantitative detection kit

  • G2026-1000T

Product Description

Product introduction:

There are two most commonly used methods for quantitative detection of protein concentration: Bradford and BCA. The basic principle of quantitative detection of proteins by BCA method is based on biuret reaction, that is the Cu2+ is reduced under alkaline conditions Cu+, and then chelating with BCA (Bicinchonic acid, a chromogenic agent). Each molecule chelates one Cu, to form a purple water-soluble complex. The substance has the highest absorption value at 562nm and the color depth is proportional to the protein concentration, so it can be used for quantitative detection of protein, Protein concentration has a good linear relationship in the range 50-2000μg/mL concentration.

This method is not affected by the chemical substances in most samples, Compatible with high concentrations of scale remover, SDS, with concentrations up to 5% included 5% of Triton X-100, 5% of Tween-20、60、80, etc. But chelating agents and high concentrations of reducing agents affect the results, Ensure no EGTA, in samples EDTA concentration below 10mM, DTT less than 1mM, β-mercaptoethanol is below 1mM. If the sample contains a chelating agent or reducing agent, Consider our other products G2001 Bradford protein quantitative detection kit.

 

Package Contents:

Cat. No.

Product description

Volume

G2026

BCA protein quantitative detection kit

1000T

 

Components:

Component Number

Component

G2026-1000T

G2026-1

BCA reagent

2×100 mL

G2026-2

Copper sulfate solution

5×1.2 mL

G2026-3

BSA

5×25 mg

G2026-4

BSA solution

5×1.5 mL

 

Storage condition:

The whole reagents kit can be transported at room temperature; protein standard (BSA) is stored at 4℃, valid for 12 months. After the protein standard solution ,stored at -20℃ and used within 6 months. The remaining reagents are stored at room temperature and valid for 12 months.

 

Usage:

1. Protein standard reserve solution: 1mL protein standard preparation solution was added to the BSA protein standard tube, and 25mg protein standard was completely dissolved, that is the protein standard reserve solution with concentration of 25mg/mL was obtained. The protein standard solution can be preserved for -20℃ for a long time.

2. Preparation of protein standard working solution: take appropriate amount of 25mg/mL protein standard reserve solution, dilute 50times with PBS or normal saline solution, get the protein standard working solution with final concentration of 0.5mg/mL. Pay attention to dilution according to 10 times gradient method to ensure accurate dilution.

3. Standard curve (enzyme labeling method): the protein standard working solution was added to 96 well plate according to 0, 1, 2, 4, 8, 12, 16, 20ul respectively, and then added 20, 19, 18, 16, 12, 8, 4, 0ul with PBS or normal saline solution, and then the gradient working solution was replenished to 20ul. Then gradient curve of protein concentration of  0, 25, 50, 100, 200, 300, 400, 500ug/mL was obtained.

4. Preparation of samples to be tested: the protein samples to be tested are properly diluted (the protein concentration of the sample can be detected in the standard curve to ensure that the test results are credible) and added to the 96 well plate according to the amount of 20μL per sample. The sample to be tested and the protein standard were diluted with the same solution.

5. Preparation of BCA chromogenic working solution: the BCA reagent and copper sulfate solution were mixed evenly according to 50:1 volume ratio to obtain the BCA chromogenic working solution. BCA chromogenic working fluid can be stored at room temperature for 24 h. Each sample to be tested needs 200μL, suggested to be prepared on demand to avoid waste.

6. Test: add 200μL, of BCA chromogenic working fluid to standard curve sample hole and sample hole to be tested Mix well (96 well plate can be placed on oscillator for 30s), 37℃ after 30 min of reaction, using standard curve 0 as reference, Colorimetry at 562 nm wavelength, Record the absorbance of each hole. (Note: reaction can also be at room temperature 2 h, or 60℃ reaction 30 min. If the protein concentration is low, it is recommended to 60℃ reaction)

7. Calculation: the standard curve is drawn with gradient protein content (μg/mL) as the horizontal coordinate and absorptivity as the vertical coordinate. According to the absorptivity of the sample, the protein concentration (μg/mL) of the sample to be tested in the corresponding hole can be found on the standard curve, and then multiplied by the dilution multiple of the sample is the actual protein concentration of the sample to be tested.

 

Notes:

1. BCA protein quantitative detection is greatly affected by temperature and time, the absorbance value will change with the prolongation of time or the increase of temperature. If the time and temperature of color reaction can not be accurately controlled, it is suggested that a standard curve should be made for each determination.

2. When preparing protein standard reserve solution, it is necessary to ensure sufficient dissolution. When diluting the standard working solution of protein, it is recommended to dilute 10 times gradient, not to dilute 50 times at a time, so as to avoid large error.

3. In order to ensure the accurate quantification of protein, it is better to choose the same buffer solution for sample extraction and dilution of protein standard, which is consistent with the conditions of detection. If the buffer itself has a high background value, it is recommended to use other methods.

4. Please wear lab clothes and disposable gloves during operation.

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