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BCA Protein Assay Kit (BCA蛋白浓度测定试剂盒) 目录号 GK10009

Bicinchoninic Acid Kit for Protein Determination

规格 价格 库存 购买数量
5mL (500 tests)
¥250.00
现货
25mL (2500 tests)
¥750.00
现货

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Sample solution is provided at 25 µL, 10mM.

实验参考方法

A. Test Tube Procedure

1.Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml of BCA Reagent A with 1 ml BCA Reagent B.
Note: Use the following formula to determine the total volume of working reagent required. (# standards+ # unknowns) x (# replicates) x (volume of working reagent per sample)= total volume of working reagent required.
2.Follow Table 1 to prepare a fresh set of standards. (Dilute Albumin (BSA) Standards with 0.9% NaCl or PBS)

Table 1: Preparation of Albumin (BSA) Standards

Tube Number Volume of Diluent (μl) Volume of BSA (μl) Final BSA Concentration (μg/ml)
A 0 μl 900 μl of 2 mg/ml Stock 2000 μg/ml
B 100 μl 300 μl of tube A 1500 μg/ml
C 300 μl 300 μl of tube A 1000 μg/ml
D 200 μl 200 μl of tube B 750 μg/ml
E 300 μl 300 μl of tube C 500 μg/ml
F 300 μl 300 μl of tube E 250 μg/ml
G 300 μl 300 μl of tube F 125 μg/ml
H 400 μl 100 μl of tube G 25 μg/ml
I 300 μl 0 0 (blank)

3.Add 0.1 ml of each standard and protein samples into separate labeled test tubes.
4.Add 2 ml of BCA working reagent to each tube and mix well.
5.Incubate at 37°C for 30 minutes.
Note: Increasing the incubation time and temperature can increase the net 562 nm absorbance for each test and decreases both minimum detection level and test range of the kit.
6.Cool all tubes to room temperature (RT).
7.Set the wavelength of spectrophotometer at OD 562 nm. Calibrate the instrument to zero by using water. Subsequently, measure the absorbance of all samples within 10 minutes.
Note: Color development continues even after cooling to RT. However, the subsequent development at RT is too weak to produce significant error if all absorbance measurements are made within 10 minute.
8.Subtract OD562 of Blank from all readings.
9.Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

B. Microplate Procedure

1. Mix BCA Reagent A and BCA Reagent B at 50:1. i.e., mix 50 ml of BCA Reagent A with 1 ml BCA Reagent B.
Note: Use the following formula to determine the total volume of working reagent required. (# standards+ # unknowns) x (# replicates) x (volume of working reagent per sample)= total volume of working reagent required.
2.Follow Table 2 to prepare a fresh set of standards. (Dilute Albumin (BSA) Standards with 0.9% NaCl or PBS)

Table 2: Preparation of Albumin (BSA) Standards

Tube Number Volume of Diluent(μl) Volume of BSA (μl) Final BSA Concentration (μg/ml)
A 0 μl 200 μl of 2 mg/ml Stock 2000 μg/ml
B 30 μl 90 μl of tube A 1500 μg/ml
C 60 μl 60 μl of tube A 1000 μg/ml
D 60 μl 60 μl of tube B 750 μg/ml
E 60 μl 60 μl of tube C 500 μg/ml
F 60 μl 60 μl of tube E 250 μg/ml
G 60 μl 60 μl of tube F 125 μg/ml
H 100 μl 25 μl of tube G 25 μg/ml
I 60 μl 0 0 (blank)

3. Add 25 μl of each standard and protein samples into separate microplate wells.
4. Add 200 μl of BCA working reagent to each well and mix well.
5. Seal plates and incubate at 37°C for 30 minutes.
6. Cool plate to room temperature (RT).
7. Measure the absorbance at 562 nm on a plate reader within 10 minutes.
8. Subtract OD562 of Blank from all readings. Plot the BSA standard curve: OD562 (on Y axis) vs BSA Standard concentration (on X axis). Use the standard curve to determine the protein concentration of each unknown sample.

Important product information

1. If this kit is received or stored cold, a precipitate may form in Reagent A or Reagent B. To dissolve the precipitate, warm the solution slowly at 37°C while mixing or microwave for a few seconds. Discard the kit if it is contaminated by bacteria
2. If interference caused by reducing substances or metal- chelating substances contained in the sample remains, Bradford Assay Kit is recommended.
3. It is recommended that the standard of different concentrations and samples be assayed in duplicate. Standard curve should be plotted for each assay.
4. Newly formed green turbidity will disappear after mixing Reagent A and Reagent B thoroughly. It will not affect the performance.
5. Assayed sample amount will be reduced while using spectrophotometer to detect protein concentration. When using 37° C incubator, prevent the influence of water evaporation.
6. Several ways for eliminating or minimizing the effects of interfering substances:
• Remove interfering substances by dialysis or gel filtration.
• Dilute the sample until substances no longer interfere.
• Since detergents of high concentrations also affect the results, precipitate the proteins in the sample with trichloroacetic acid (TCA).
7.Avoid using substances including reducing substances, chelating agents, strong acid and alkali since they interfere with protein estimation even at very low concentration.

产品组成

Components 500 tests 2500 tests 5000 tests
BCA Reagent A 100 mL 500 mL 500 mL × 2
BCA Reagent B 3 mL 5 mL × 3 5 mL × 6
Albumin (BSA) Standards
5mg/mL
1 mL 5 mL 5 mL × 2

功能属性

应用Western blotting
Protein expression assays
Protein profiling and characterization
Protein quantitation assays
运输方式Ship with blue ice.
储存条件BCA Reagent A and B store at room temperature.It is stable at room temperature for one year. Albumin (BSA) Standards store at -20°C.
用途仅供研究使用!不能用于人体。

产品描述

BCA Protein Assay Kit is a ready-to-use detergent- compatible Western blot related total protein analysis reagent used for the quick determination of total protein concentration by measuring absorbance at 562 nm and comparing to a protein standard absorption vs. concentration curve, according to Smith. The protein quantification process can be finished in 45 minutes.
The BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantization of total protein. This method combines the well- known reduction of Cu2+ to Cu1+ by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) using a unique reagent containing bicinchoninic acid. The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 μg/ml).
The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA. Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color producing functional groups.
Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown before the concentration of each unknown is determined based on the standard curve. If precise quantization of an unknown protein is required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples. Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1 ml) of protein sample; however, because it uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized.