Bradford Protein Colorimetric Assay Kit
SKU: E-BC-K168-M-500
Bradford Protein Colorimetric Assay Kit
SKU # | E-BC-K168-M |
Detection Instrument | Microplate reader (550 nm-630 nm, optimum wavelength: 595 nm) |
Detection method | Colorimetric method |
Product Details
Properties
Synonyms | TP |
Sample Type | Serum, plasma, animal tissue and cell |
Sensitivity | 0.046 mg/mL |
Detection Range | 0.046-0.6 mg/mL |
Detection Method | Colorimetric method |
Assay type | Quantitative |
Assay time | 20 min |
Precision | Average inter-assay CV: 8.200% | Average intra-assay CV: 3.200% |
Other instruments required | Micropipettor, Incubator, Vortex mixer, Centrifuge |
Other reagents required | Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4) |
Storage | Reagent 2: RT, Reagent 1: 2-8℃ |
Valid period | 12 months |
Dilution of Sample
It is recommended to take 2~3 samples with expected large difference to do pre-experiment before formal experiment and dilute the sample according to the result of the pre-experiment and the detection range ( MERGEFIELD Detection_range 0.046-0.6 mg/mL).
The recommended dilution factor for different samples is as follows (for reference only):
Sample type | Dilution factor |
Human serum | 90-110 |
Human plasma | 90-110 |
Rabbit serum | 90-110 |
Rat plasma | 90-110 |
Chicken serum | 90-110 |
10% Mouse kidney tissue homogenate | 15-20 |
10% Mouse lung tissue homogenate | 15-20 |
10% Rat spleen tissue homogenate | 15-20 |
10% Rat heart tissue homogenate | 15-20 |
10% Rat liver tissue homogenate | 20-25 |
293T | 1 |
HL-60 | 1 |
Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).
Detection Principle
Coomassie brilliant blue G-250 is red under the free state, and it has the maximum absorbance at 465 nm. When the Coomassie brilliant blue G-250 combined to protein, the compound will have the maximum at 595 nm. The absorbance value is directly proportional to the protein content, so the concentration of total protein can be calculated directly by measuring the OD value at 595 nm.
Kit Components & Storage
Item | Component | Size 1 (96 T) | Size 2 (500 Assays) | Storage |
Reagent 1 | Chromogenic Agent Stock Solution | 6 mL × 1 vial | 30 mL × 1 vial | 2-8°C, 12 months, shading light |
Reagent 2 | 1 mg Standard | 1 mg × 2 vials | 1 mg × 5 vials | RT, 12 months |
Microplate | 96 wells | |||
Plate Sealer | 2 pieces |
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three human serum samples were assayed in replicates of 20 to determine precision within an assay. (CV = Coefficient of Variation)
Parameters | Sample 1 | Sample 2 | Sample 3 |
Mean (mg/mL) | 0.10 | 0.24 | 0.52 |
%CV | 3.5 | 3.1 | 3.0 |
Inter-assay Precision
Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.
Parameters | Sample 1 | Sample 2 | Sample 3 |
Mean (mg/mL) | 0.10 | 0.24 | 0.52 |
%CV | 8.1 | 7.4 | 9.1 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 104%.
Standard 1 | Standard 2 | Standard 3 | |
Expected Conc. (mg/mL) | 0.08 | 0.24 | 0.57 |
Observed Conc. (mg/mL) | 0.1 | 0.3 | 0.6 |
Recovery rate (%) | 101 | 106 | 102 |
Sensitivity
The analytical sensitivity of the assay is 0.046 mg/mL. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.
Standard Curve
As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only.
Concentration (mg/mL) | 0 | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.6 |
OD value | 0.320 | 0.337 | 0.358 | 0.405 | 0.452 | 0.502 | 0.528 | 0.568 |
0.321 | 0.332 | 0.373 | 0.413 | 0.458 | 0.484 | 0.542 | 0.571 | |
Average OD | 0.320 | 0.335 | 0.366 | 0.409 | 0.455 | 0.493 | 0.535 | 0.569 |
Absoluted OD | 0.000 | 0.015 | 0.046 | 0.089 | 0.135 | 0.173 | 0.215 | 0.249 |