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BCA Protein Colorimetric Assay Kit
SKU: E-BC-K318-M-500
BCA Protein Colorimetric Assay Kit
| SKU # | E-BC-K318-M |
| Detection Instrument | Microplate reader (540-590 nm, optimum wavelength: 562 nm) |
| Detection method | Colorimetric method |
Product Details
Properties
| Synonyms | TP |
| Sample type | Serum, plasma, cell culture supernatant, tissue, cells |
| Sensitivity | 0.0165 mg/mL |
| Detection range | 0.0165-1 mg/mL |
| Detection Method | Colorimetric method |
| Assay type | Quantitative |
| Assay time | 40 min |
| Precision | Average inter-assay CV: 4.500% | Average intra-assay CV: 2.200% |
| Other instruments required | Vortex mixer, Micropipettor, Incubator |
| Other reagents required |
Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4) |
| Storage | RT |
| 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 (0.0165-1 mg/mL).
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| 10% Mouse brain tissue homogenization | 8-12 |
| 10% Mouse kidney tissue homogenization | 8-12 |
| Human serum | 100-200 |
| 10% Rat liver tissue homogenization | 15-20 |
| 10% Mouse heart tissue homogenization | 8-12 |
| Rat serum | 100-200 |
Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).
Detection Principle
Cu2+ can be reduced to Cu+ by protein in alkaline condition. Cu+ can combine with BCA reagent and form purple complex, which has a maximum absorption peak at 562 nm. The absorbance value is proportional to the protein concentration. Therefore, the protein concentration can be calculated according to the OD value.
Kit Components & Storage
| Item | Component | Size 1 (96 T) |
Size 2 (500 Assays) |
Storage |
| Reagent 1 | BCA Reagent | 25 mL × 1 vial | 50 mL × 2 vials | RT, 12 months |
| Reagent 2 | Copper Salt Solution | 0.5 mL × 1 vial | 3 mL × 1 vial | RT, 12 months |
| Reagent 3 | Standard | 1 mg × 1 vial | 1 mg × 5 vials | RT, 12 months |
| Reagent 4 | Standard Diluent | 15 mL × 1 vial | 30 mL × 1 vial | RT, 12 months |
| Microplate | 96 wells | No requirement | ||
| 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) | 0.08 | 0.28 | 0.65 |
| %CV | 2.5 | 2.3 | 1.8 |
Inter-assay Precision
Three human serum samples were assayed 17 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (mg/mL) | 0.08 | 0.28 | 0.65 |
| %CV | 4.2 | 4.5 | 4.8 |
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 100%.
| Parameters | Standard 1 | Standard 2 | Standard 3 |
| Expected Conc. (mg/mL) | 0.25 | 0.45 | 0.85 |
| Observed Conc. (mg/mL) | 0.3 | 0.4 | 0.9 |
| Recovery rate (%) | 101 | 99 | 100 |
Sensitivity
The analytical sensitivity of the assay is 0.0165 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 | 0.2 | 0.3 | 0.4 | 0.6 | 0.7 | 0.9 | 1 |
| Average OD | 0.081 | 0.308 | 0.395 | 0.494 | 0.666 | 0.761 | 0.906 | 0.984 |
| Absoluted OD | 0 | 0.227 | 0.314 | 0.413 | 0.585 | 0.680 | 0.825 | 0.903 |