BCA Protein Quantification Kit

• C0050-Instruction-Manual(823 KB)

1.High Sensitivity: detects protein quantity down to 0.5 μg and concentration down to 25 μg/mL. Can test sample volume ranging from 1 to 20 μL.

2.Wide assay range: linear working range for BSA of 25 to 2000 μg/mL.

3.Short assay time: 45-min incubation; much easier and four times faster than classical Lowry methods.

4.Good Compatibility: unaffected by typical concentrations of most detergents and compatible with up to 5% SDS, 5% Triton X-100, and 5% Tween 20, 60, 80 in samples.

Note: please ensure no EGTA is present, EDTA is below 10 mM, DTT is below 1 mM, and β-Mercaptoethanol is below 0.01%.

1. Prepare the protein standards and samples

1)Add 1.2 mL of preparation solution to 30 mg of BSA protein standards, and dissolve thoroughly to prepare a 25 mg/mL standard solution. Store the solution at -20°C.

2)Take an appropriate amount of the 25 mg/mL standard solution and dilute it to a final concentration of 0.5 mg/mL. This solution can be stored long-term at -20°C.

Note: Use the solvent of the protein sample as the protein diluent. 0.9% NaCl or PBS are also options for the protein diluent.

3)To create gradient concentration in a 96-well plate, dilute the protein standard solution by the following amount in each well.

4)The final volume of each sample well should be 20 μL.

2. Prepare BCA Working Reagent

Prepare BCA working reagent by mixing 50 parts of Reagent A with 1 part of Reagent B (50:1, Reagent A:B). For the above example, combine 5 mL of Reagent A with 100 μL of Reagent B and mix thoroughly to prepare 5.1 mL of BCA working reagent. The BCA working reagent is stable at room temperature for 24 hours.

3. Determine the concentration of protein

1)Add 200 μL of BCA working reagent to each well and incubate at 37 °C for 20-30 minutes.

Note: Alternatively, incubate at 60 ℃ for 30 minutes or at room temperatuure for 2 hours. The color will darken over time and the color development will continue with increasing temperature. For low concentrations, incubate at a higher temperature or extend the incubation time appropriately. Maintain accurate timing and temperature to ensure precise quantitation.

2)Measure the absorbance of the solution at 562 nm on a microplate reader.

3)Prepare a standard curve based on the absorbance of BSA standards (X-axis: protein concentration in μg/mL; Y-axis: A562). Determine the protein concentration in each sample based on the standard curve and the dilution factor.

• For Reagent A , Reagent B and standard solution, store at room temperature.

• For BSA protein standards , store the powder form at room temperature, and the solution form at -20°C for up to 12 months.

1.The BCA assay can be adapted for use in 96 well plates with a microplate reader. If a microplate reader is not available, a standard spectrometer can be used. However, the volumes of the BCA working reagent, standards and samples should be appropriately increased based on the minimum detection volume of the cuvette.

2.If precipitation is observed in Reagent A or Reagent B when storing at low temperature or for extended periods, incubate and agitate at 37°C until fully dissolved. Discard if bacterial contamination is detected.

3.Prepare a standard curve for each measurement as the color development can vary with temperature and time.

4.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

5.Please wear a lab coat and disposable gloves.