Before you start
- Dilute samples so they fall within the standard range (RIPA lysates are often diluted 1:5–1:10).
Reagents and materials
Totals are for 40 wells (+10% extra). Per-well amounts are in the middle column.
| Reagent | Per well | Total |
|---|---|---|
| Standard or sample | 25 µL | 25 µL |
| BCA working reagent (50 parts A : 1 part B) | 200 µL | 200 µL |
Buffers and recipes
BSA standard series (µg/mL) per plate
| A | 2000 |
| B | 1500 |
| C | 1000 |
| D | 750 |
| E | 500 |
| F | 250 |
| G | 125 |
| H | 25 |
| Blank | 0 (lysis buffer only) |
Prepare standards in the same buffer as the samples.
Equipment
- 96-well plate
- 37 °C incubator
- Plate reader at 562 nm
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Prepare standards
Make the BSA series in the same buffer as your samples.
Load
Pipette 25 µL standards and samples in duplicate or triplicate.
Add working reagent
Add 200 µL working reagent and mix on a plate shaker for 30 s.
Incubate
Cover and incubate at 37 °C for 30 min.
Read
Cool to room temperature and read at 562 nm.
Calculate
Subtract the blank, fit the standard curve (linear or quadratic) and calculate sample concentrations × dilution factor.
Critical points
- Reducing agents (DTT, β-mercaptoethanol) and chelators interfere with BCA; use a compatible kit or Bradford.
- Colour keeps developing slowly — read all wells at the same time.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Samples above range | Dilute further and repeat. |
| Purple blank | Interfering substance in the buffer; dilute or use a compatible assay. |
Safety
Reagent A is alkaline; reagent B contains copper sulfate.
Further reading
- Smith PK et al. Measurement of protein using bicinchoninic acid. Anal Biochem (1985).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.