The BCA method is one of the most widely used techniques for protein quantification. This kit, based on the bicinchoninic acid (BCA) assay, enables rapid, stable, and sensitive measurement of protein concentrations. Its principle relies on the reduction of Cu²⁺ to Cu⁺ by peptide bonds in an alkaline environment, followed by the formation of a purple-colored complex between Cu⁺ and BCA. This complex exhibits strong absorbance at 562 nm, proportional to protein concentration. The kit includes a series of ready-to-use protein standard solutions (BSA), eliminating the need for dilution and simplifying the workflow.
Product Component List:
R1491648
| Component
| 500T
| 500T×5
| Storage
|
R1491648A
| Reagent A
| 100 mL | 100 mL×5
| 2-8℃ |
R1491648B
| Reagent B
| 3 mL | 3 mL×5
| 2-8℃
|
R1491648C
| Ready-to-use BSA standard① (0 μg/mL) | 1 mL | 1 mL×5
| -20℃ |
R1491648D
| Ready-to-use BSA standard② (125 μg/mL)
| 1 mL
| 1 mL×5
| -20℃
|
R1491648E
| Ready-to-use BSA standard③ (250 μg/mL)
| 1 mL
| 1 mL×5
| -20℃
|
R1491648F
| Ready-to-use BSA standard④ (500 μg/mL)
| 1 mL
| 1 mL×5
| -20℃
|
R1491648G
| Ready-to-use BSA standard⑤ (750 μg/mL)
| 1 mL
| 1 mL×5
| -20℃
|
R1491648H
| Ready-to-use BSA standard⑥ (1000 μg/mL)
| 1 mL
| 1 mL×5
| -20℃
|
R1491648I
| Ready-to-use BSA standard⑦ (1500 μg/mL)
| 1 mL
| 1 mL×5
| -20℃
|
R1491648J
| Ready-to-use BSA standard⑧ (2000 μg/mL)
| 1 mL
| 1 mL×5
| -20℃ |
Key Features
Convenient: Ready-to-use standards avoid tedious dilution steps.
High Accuracy: Lower coefficient of variation than Coomassie Brilliant Blue assays.
Broad Linear Range: Detection range: 20–2000 μg/mL.
Excellent Compatibility: Tolerates metal ions, reducing agents, chelators, and detergents.
Protocol (Microplate Reader Example)
Prepare Working Reagent:
a. Calculate the total volume required:
Working reagent volume = (Number of standard samples + Number of test samples) × Replicates × Volume per well
Example: 8 standards + 3 test samples with 3 replicates each at 200 μL/well:
(8 + 3) × 3 × 200 μL = 6.6 mL
b. Mix Reagent A and Reagent B at a 50:1 ratio to prepare the working reagent. Vortex thoroughly.
Note:
Quantification Steps:① Add 20 μL of each ready-to-use BSA standard (①–⑧) to a 96-well plate (mix thoroughly before use):
Well
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Standard
| ① | ②
| ③
| ④
| ⑤
| ⑥
| ⑦
| ⑧
|
Volume (μL)
| 20 | 20
| 20
| 20
| 20
| 20
| 20
| 20
|
BSA (μg/mL)
| 0 | 125 | 250 | 500 | 750 | 1000 | 1500 | 2000 |
② Dilute test samples in 1× PBS or 0.9% saline (e.g., 2×, 4×, 8× serial dilutions). Add 20 μL to wells.
③ Add 200 μL working reagent to each well. Mix thoroughly, cover the plate, and incubate at 37°C for 30 min. Cool to room temperature.
Note: Incubation alternatives:
④ Measure absorbance at 562 nm (or 540–590 nm) using a microplate reader. Subtract the blank control (Standard ① + working reagent) absorbance.
⑤ Plot a standard curve and calculate sample protein concentrations.
Note:
Exclude obvious outliers.
Multiply calculated concentrations by dilution factors.
Use linear regression for computer-based fitting.
Precautions
Compatible with microplate readers (microplate method) or spectrophotometers (cuvette method). For cuvettes, ensure the volume meets the minimum requirement (may reduce total number of assays).
If precipitates form during cold/long-term storage, stir or warm to 37°C to dissolve.
Always generate a fresh standard curve for each assay to ensure accuracy.
For samples with high levels of interfering substances (see Appendix), consider alternative protein quantification methods.
Wear a lab coat and disposable gloves during operations.
For research use only.
G2601309