BCA Protein Concentration Assay Kit Product Description: BCA protein quantification method is a rapid, sensitive, stable and reliable method for the quantitative determination of proteins. The measurement range is 10-2000 ug/ml, which is superior to the Lowry method for detecting total protein content. The product. BCA
The method determines the protein concentration from the chemicals in most samples. It has the largest absorption at 562 nm, and the concentration of the protein to be tested can be calculated by comparison with a standard curve.
Principle: BCA (bicinchonininc acid) and other reagents such as copper sulfate ion copper sulfate, mixed together to show apple green, that is, BCA working reagent. Under alkaline conditions, when BCA binds to protein, the protein reduces Cu2+ to Cu+, and one Cu+ sequesters two BCA molecules. The working reagent forms a purple complex from the original apple green, and the maximum light absorption intensity is proportional to the protein concentration.
Kit composition:
BCA01 (50ml) BCA02 (100 ml)
Reagent A: BCA alkaline solution 50ml 100ml
Reagent B: copper sulfate solution 1ml 2 ml
Reagent C: Standard BSA protein (1 mg/ml) 1 ml 1 ml × 2
1.5 ml centrifuge tube 25 50 instructions One kit storage: Reagents A and B have a stable period of at least 1 year at room temperature. Standard protein solution is stored at -20 ° C,
Short-term storage at 4 ° C.
Determination method 1: (test tube method)
1. Prepare the working fluid: According to the standard product and the sample quantity, prepare an appropriate amount of BCA working solution according to 50 volumes of reagent A and 1 volume of reagent B, and mix well. Note: BCA working fluid is stable for 24 hours at room temperature. The amount of working fluid to be formulated corresponds to the cuvette used for the measurement. Two to three parallel reactions are required for each assay. The colorimetric system listed in this section is used.
A 0.5 ml cuvette; if there is no 0.5 ml cuvette, the reaction system needs to be enlarged to the volume required for the exact reading of the cuvette that will be used in the experiment.
2. Preparation of BSA standards and samples: The sample is mixed with water or other buffering configuration that does not interfere with the color reaction, so that the concentration to be determined is in the linear portion of the standard curve. Three replicates were prepared for each reaction. Standard curve is generally 5-6
The points can be determined, and the specific concentration of each point is determined according to the estimated concentration of the sample. When diluting BSA, use water or a solution consistent with the sample. If the concentration to be measured is about 200 μg/ml, add the BSA standard, sample and
BCA working fluid.
3. Take an appropriate volume of standard protein (protein sample) and mix it with protein solution: working solution at a ratio of 1:20.
The mixture was incubated at 37 ° C for 30 min and cooled to room temperature.
4. Measure the absorbance of the sample and the standard at 562 nm.
1. Draw a standard curve and calculate the concentration of the sample through the standard curve. Note: The actual concentration needs to be multiplied by the dilution factor of the sample.
1 2 3 4 5 6 7 Dilution of sample to be tested 1
Sample to be tested diluted 2
BSA (μl) 0 2.5 5 10 15 20 25
H2O (μl) 25 22.5 20 15 10 5 0 0 0
Working fluid (μl) 500 500 500 500 500 500 500 500 500
OD562
OD562 average method 2: (96-well plate)
1. Prepare BCA working solution: According to the standard product and sample quantity, prepare appropriate amount of BCA working solution according to 50 volumes of reagent A and 1 volume of reagent B, and mix well.
2. Add 0, 1, 2, 4, 6, 8, 10 microliters of protein standard to the wells of the 96-well plate protein standard, add sterilized double distilled water to make up 10 μl; take 10 μl The sample to be tested is added to a 96-well plate. Each measurement should be done 2-3 parallels.
3. Add 200 μl of BCA to the sample well and the protein standard well.
Mix the working solution (ie, the volume ratio of the sample to the working fluid is 1:20).
4, 37 ° C warm bath for 30 min. Cold to room temperature.
5. The absorbance was measured at a wavelength of 562 nm by a microplate reader.
6. Make a standard curve and determine the sample concentration from the standard curve.
Precautions:
1. The depth of the reaction color is related to the temperature of the reaction, in addition to the protein concentration of the sample. If the concentration of the sample is high (>50μg/ml), the reaction temperature is generally 37 degrees. If the concentration of the sample protein is low (<50 μg), the reaction temperature is 60 ° C. At this temperature, tryptophan, tyrosine and peptide bonds are sufficiently oxidized, greatly improving the detection sensitivity.
2. The protein concentration detected by this method ranges from 20μg to 500μg/ml; when the protein concentration is <20ug/ml, 60°C is recommended.
Warm the bath and adjust the ratio of protein solution: working solution to 1:8 (enhancement method) to detect 5 ug/ml.
Reagents A and B have a stability period of at least 1 year at room temperature. The standard protein solution is stored at -20 ° C and stored at 4 ° C for short periods.
The method determines the protein concentration from the chemicals in most samples. It has the largest absorption at 562 nm, and the concentration of the protein to be tested can be calculated by comparison with a standard curve.
Principle: BCA (bicinchonininc acid) and other reagents such as copper sulfate ion copper sulfate, mixed together to show apple green, that is, BCA working reagent. Under alkaline conditions, when BCA binds to protein, the protein reduces Cu2+ to Cu+, and one Cu+ sequesters two BCA molecules. The working reagent forms a purple complex from the original apple green, and the maximum light absorption intensity is proportional to the protein concentration.
Kit composition:
BCA01 (50ml) BCA02 (100 ml)
Reagent A: BCA alkaline solution 50ml 100ml
Reagent B: copper sulfate solution 1ml 2 ml
Reagent C: Standard BSA protein (1 mg/ml) 1 ml 1 ml × 2
1.5 ml centrifuge tube 25 50 instructions One kit storage: Reagents A and B have a stable period of at least 1 year at room temperature. Standard protein solution is stored at -20 ° C,
Short-term storage at 4 ° C.
Determination method 1: (test tube method)
1. Prepare the working fluid: According to the standard product and the sample quantity, prepare an appropriate amount of BCA working solution according to 50 volumes of reagent A and 1 volume of reagent B, and mix well. Note: BCA working fluid is stable for 24 hours at room temperature. The amount of working fluid to be formulated corresponds to the cuvette used for the measurement. Two to three parallel reactions are required for each assay. The colorimetric system listed in this section is used.
A 0.5 ml cuvette; if there is no 0.5 ml cuvette, the reaction system needs to be enlarged to the volume required for the exact reading of the cuvette that will be used in the experiment.
2. Preparation of BSA standards and samples: The sample is mixed with water or other buffering configuration that does not interfere with the color reaction, so that the concentration to be determined is in the linear portion of the standard curve. Three replicates were prepared for each reaction. Standard curve is generally 5-6
The points can be determined, and the specific concentration of each point is determined according to the estimated concentration of the sample. When diluting BSA, use water or a solution consistent with the sample. If the concentration to be measured is about 200 μg/ml, add the BSA standard, sample and
BCA working fluid.
3. Take an appropriate volume of standard protein (protein sample) and mix it with protein solution: working solution at a ratio of 1:20.
The mixture was incubated at 37 ° C for 30 min and cooled to room temperature.
4. Measure the absorbance of the sample and the standard at 562 nm.
1. Draw a standard curve and calculate the concentration of the sample through the standard curve. Note: The actual concentration needs to be multiplied by the dilution factor of the sample.
1 2 3 4 5 6 7 Dilution of sample to be tested 1
Sample to be tested diluted 2
BSA (μl) 0 2.5 5 10 15 20 25
H2O (μl) 25 22.5 20 15 10 5 0 0 0
Working fluid (μl) 500 500 500 500 500 500 500 500 500
OD562
OD562 average method 2: (96-well plate)
1. Prepare BCA working solution: According to the standard product and sample quantity, prepare appropriate amount of BCA working solution according to 50 volumes of reagent A and 1 volume of reagent B, and mix well.
2. Add 0, 1, 2, 4, 6, 8, 10 microliters of protein standard to the wells of the 96-well plate protein standard, add sterilized double distilled water to make up 10 μl; take 10 μl The sample to be tested is added to a 96-well plate. Each measurement should be done 2-3 parallels.
3. Add 200 μl of BCA to the sample well and the protein standard well.
Mix the working solution (ie, the volume ratio of the sample to the working fluid is 1:20).
4, 37 ° C warm bath for 30 min. Cold to room temperature.
5. The absorbance was measured at a wavelength of 562 nm by a microplate reader.
6. Make a standard curve and determine the sample concentration from the standard curve.
Precautions:
1. The depth of the reaction color is related to the temperature of the reaction, in addition to the protein concentration of the sample. If the concentration of the sample is high (>50μg/ml), the reaction temperature is generally 37 degrees. If the concentration of the sample protein is low (<50 μg), the reaction temperature is 60 ° C. At this temperature, tryptophan, tyrosine and peptide bonds are sufficiently oxidized, greatly improving the detection sensitivity.
2. The protein concentration detected by this method ranges from 20μg to 500μg/ml; when the protein concentration is <20ug/ml, 60°C is recommended.
Warm the bath and adjust the ratio of protein solution: working solution to 1:8 (enhancement method) to detect 5 ug/ml.
Reagents A and B have a stability period of at least 1 year at room temperature. The standard protein solution is stored at -20 ° C and stored at 4 ° C for short periods.
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