BCA protein concentration determination kit
BCA Protein Assay Kit â— Kit contents and storage:
â— Storage conditions and validity period:
Reagent A and reagent B were stored at room temperature; bovine serum albumin standard solution was stored at -20 ° C; PBS solution was stored at 4 ° C. This kit is valid for 1 year.
â— Product introduction:
The principle of the BCA protein concentration determination kit is that the peptide bond structure in the protein molecule can form a complex with Cu 2+ in an alkaline environment, and reduce Cu 2+ to Cu + , and the BCA reagent can specifically interact with Cu + . The combination forms a stable colored complex and has a maximum light absorption at 562 nm. The color of the complex is proportional to the protein concentration, and the protein content can be determined according to the amount of absorption.
This kit can detect 500 samples (using microplates) or 50 samples (using test tubes).
â— Features:
1. High sensitivity, the lower limit of detection concentration reaches 25μg/ml (there is a good linear relationship in the concentration range of 20-1000μg/ml), the minimum detection protein amount is 0.2μg, and the sample volume to be tested is 1-20μl.
2. The biggest advantage of the BCA method for determining protein concentration is that the protein concentration can be measured to withstand high concentrations of detergent, compatible with up to 5% SDS in samples, 5% Triton X-100, 5% Tween 20, 60 , 80. However, due to the chelating agent and a slightly higher concentration of reducing agent, it is necessary to ensure that EDTA is less than 10 mM, no EGTA, dithiothreitol is less than 1 mM, and β-mercaptoethanol is less than 0.01%.
â— Operation method
BCA working fluid preparation:
Reagent A and reagent B were mixed at a volume ratio of 50:1 to prepare a BCA working solution.
For example, 50 ml of reagent A is mixed with 1 ml of reagent B to prepare 51 ml of BCA working solution.
Note: BCA working fluid can be left at room temperature for one week without failure.
Microplate assay procedure: (working range 20-2000 μg/ml )
1. Preparation of protein standard: completely dissolve the protein standard at room temperature, and take 20 μl of 5 mg/ml BSA protein standard solution and dilute to 100 μl with PBS solution to a final concentration of 1.0 mg/ml.
2. Prepare the BSA standard assay solution according to the following table:
3. Add the appropriate volume of the sample to be tested to the microplate and make up to 20 μl with PBS.
4. Add 200 μl of BCA working solution to the microplate, mix and place at 37 ° C for 30 minutes;
Note: It can also be placed at room temperature for 2 hours or at 60 ° C for 30 minutes. When the protein concentration is determined by the BCA method, the color is deepened with time. And the color reaction will increase as the temperature increases. If the concentration is low, it is suitable to incubate at a higher temperature, or to extend the incubation time appropriately.
5. Determine the absorbance at 562 nm and record the reading; use the absorbance of the BSA-free sample as a blank.
6. With A 562 as the ordinate and BSA as the abscissa, draw a standard curve and calculate the protein concentration in the sample. If the resulting protein concentration is not within the standard curve, dilute the sample and re-measure.
Test tube test procedure: (working range 20-1000 μg/ml )
1. Preparation of protein standard: Completely dissolve the protein standard at room temperature, take 150 μl of 5 mg/ml BSA protein standard solution, and add 600 μl of PBS solution to 750 μl to a final concentration of 1.0 mg/ml.
2. Prepare the BSA standard assay solution according to the following table:
3. Add the appropriate volume of the sample to be tested to the test tube and make up to 100 μl with PBS;
4. Add 2 ml of BCA working solution to the test tube, mix and place at 37 ° C for 30 minutes;
Note: It can also be placed at room temperature for 2 hours or at 60 ° C for 30 minutes. When the protein concentration is determined by the BCA method, the color is deepened with time. And the color reaction will increase as the temperature increases. If the concentration is low, it is suitable to incubate at a higher temperature, or to extend the incubation time appropriately.
5. Determine the absorbance at 562 nm and record the reading; use the absorbance of the BSA-free sample as a blank.
6. With A 562 as the ordinate and BSA as the abscissa, draw a standard curve and calculate the protein concentration in the sample. If the resulting protein concentration is not within the standard curve, dilute the sample and re-measure.
â— References:
1. Smith, PK, et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem . 150 :76-85.
2. Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal Biochem . 175 :231-7.
3. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem . 159 :138-42.
4. Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem . 180 :136-9.
BCA Protein Assay Kit â— Kit contents and storage:
| product name | package | Storage method |
| BCA Reagent A | 100 ml | RT |
| BCA Reagent B | 3 ml | RT |
| Bovine serum albumin (BSA) standard solution (5 mg/ml) | 2×1 ml | -20 ° C |
| PBS solution | 10 ml | 4 ° C |
| Instruction manual | 1 serving |
Reagent A and reagent B were stored at room temperature; bovine serum albumin standard solution was stored at -20 ° C; PBS solution was stored at 4 ° C. This kit is valid for 1 year.
â— Product introduction:
The principle of the BCA protein concentration determination kit is that the peptide bond structure in the protein molecule can form a complex with Cu 2+ in an alkaline environment, and reduce Cu 2+ to Cu + , and the BCA reagent can specifically interact with Cu + . The combination forms a stable colored complex and has a maximum light absorption at 562 nm. The color of the complex is proportional to the protein concentration, and the protein content can be determined according to the amount of absorption.
This kit can detect 500 samples (using microplates) or 50 samples (using test tubes).
â— Features:
1. High sensitivity, the lower limit of detection concentration reaches 25μg/ml (there is a good linear relationship in the concentration range of 20-1000μg/ml), the minimum detection protein amount is 0.2μg, and the sample volume to be tested is 1-20μl.
2. The biggest advantage of the BCA method for determining protein concentration is that the protein concentration can be measured to withstand high concentrations of detergent, compatible with up to 5% SDS in samples, 5% Triton X-100, 5% Tween 20, 60 , 80. However, due to the chelating agent and a slightly higher concentration of reducing agent, it is necessary to ensure that EDTA is less than 10 mM, no EGTA, dithiothreitol is less than 1 mM, and β-mercaptoethanol is less than 0.01%.
â— Operation method
BCA working fluid preparation:
Reagent A and reagent B were mixed at a volume ratio of 50:1 to prepare a BCA working solution.
For example, 50 ml of reagent A is mixed with 1 ml of reagent B to prepare 51 ml of BCA working solution.
Note: BCA working fluid can be left at room temperature for one week without failure.
Microplate assay procedure: (working range 20-2000 μg/ml )
1. Preparation of protein standard: completely dissolve the protein standard at room temperature, and take 20 μl of 5 mg/ml BSA protein standard solution and dilute to 100 μl with PBS solution to a final concentration of 1.0 mg/ml.
2. Prepare the BSA standard assay solution according to the following table:
| Numbering | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 1 mg/ml BSA standard solution μl | 5 mg/ml BSA standard solution μl | ||||||||
| BSA standard solution μl | 0 | 0.5 | 2.5 | 5.0 | 10 | 15 | 20 | 6 | 8 |
| PBS solution μl | 20 | 19.5 | 17.5 | 15 | 10 | 5 | 0 | 14 | 12 |
| Final concentration of BSA μg/ml | 0 | 25 | 125 | 250 | 500 | 750 | 1000 | 1500 | 2000 |
| Total volume μl | 20 μl | ||||||||
4. Add 200 μl of BCA working solution to the microplate, mix and place at 37 ° C for 30 minutes;
Note: It can also be placed at room temperature for 2 hours or at 60 ° C for 30 minutes. When the protein concentration is determined by the BCA method, the color is deepened with time. And the color reaction will increase as the temperature increases. If the concentration is low, it is suitable to incubate at a higher temperature, or to extend the incubation time appropriately.
5. Determine the absorbance at 562 nm and record the reading; use the absorbance of the BSA-free sample as a blank.
6. With A 562 as the ordinate and BSA as the abscissa, draw a standard curve and calculate the protein concentration in the sample. If the resulting protein concentration is not within the standard curve, dilute the sample and re-measure.
Test tube test procedure: (working range 20-1000 μg/ml )
1. Preparation of protein standard: Completely dissolve the protein standard at room temperature, take 150 μl of 5 mg/ml BSA protein standard solution, and add 600 μl of PBS solution to 750 μl to a final concentration of 1.0 mg/ml.
2. Prepare the BSA standard assay solution according to the following table:
| Numbering | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 1 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 |
| Final concentration of BSA μg/ml | 0 | 25 | 125 | 250 | 500 | 750 | 1000 | 1500 | 2000 |
| Total volume μl | 100 μl | ||||||||
4. Add 2 ml of BCA working solution to the test tube, mix and place at 37 ° C for 30 minutes;
Note: It can also be placed at room temperature for 2 hours or at 60 ° C for 30 minutes. When the protein concentration is determined by the BCA method, the color is deepened with time. And the color reaction will increase as the temperature increases. If the concentration is low, it is suitable to incubate at a higher temperature, or to extend the incubation time appropriately.
5. Determine the absorbance at 562 nm and record the reading; use the absorbance of the BSA-free sample as a blank.
6. With A 562 as the ordinate and BSA as the abscissa, draw a standard curve and calculate the protein concentration in the sample. If the resulting protein concentration is not within the standard curve, dilute the sample and re-measure.
â— References:
1. Smith, PK, et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem . 150 :76-85.
2. Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal Biochem . 175 :231-7.
3. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem . 159 :138-42.
4. Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem . 180 :136-9.
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