BCA Protein Assay Kit

Cat: PC0020

Size: 500T (Microplate Reader) / 50T (Spectrophotometer)

Storage: BSA protein standard is stored at 4℃ for 3 months， or at -20℃ for 1 year, other reagents keep at room temperature for 1 year

Components:

Product Description:

• The BCA Protein Assay Kit principle:
  • Based on the reduction of Cu^2+ to Cu^+ by proteins.
  • BCA forms a purple-blue complex with Cu^+ under alkaline conditions.
  • Absorbance is detected at 562nm.
  • Protein quantification is done by comparing absorbance with a standard curve.
• Compatibility with detergents:
  • Common detergents like SDS, Triton X-100, and Tween do not affect test results.
• Sensitivity to certain agents:
  • Affected by chelating agents (EDTA, EGTA), reducing agents (DTT, mercaptoethanol), and lipids.
• High background considerations:
  • In cases of high background due to sample dilution or lysate, consider using the Bradford Protein Assay Kit.

Protocol

A. MicroplateReader

1. BCA working solution

Dilute 1 volume of Cu Reagent with 50 volumes of BCA Reagent (1:50). If precipitation occurs, it will disappear after mixing. The BCA working reagent could be kept at RT for 24 hours.

2. BSA standard working solution

Dilute 10μl BSA (5mg/ml) with PBS until 100μl (0.5mg/ml). Add 0, 2, 4, 6, 8, 12, 16, 20μl BSA standard working solution (0.5mg/ml) to 96 well plates, add PBS until to 20μl.

3. Dilute the sample properly. It is better to do several gradients, such as 2, 4, and 8 times dilution. Add 20μl sample to 96-well

Note: To avoid errors in operation steps, make the sample point located at 1/2 behind the standard line as much as possible, as the pipette has a large error when taking a small amount of sample, and the point in front of the standard line may not be very accurate.

4. BCA reaction

Add 200μl BCA working solution to 96 well plates, and incubate at 37℃ for 15-30min. Measure

the absorbance at 562nm, and draw a standard curve to calculate the sample protein content. Avoid water evaporation from affecting the test results when using an incubator.

B. Spectrophotometer

1. BCA working solution

Dilute 1 volume of Cu Reagent with 50 volumes of BCA Reagent (1:50). If precipitation occurs, it will disappear after mixing. The BCA working reagent could be kept at RT for 24 hours.

2. BSA standard working solution

Dilute 100μl BSA (5mg/ml) with PBS until 1 ml (0.5mg/ml).

3. Prepare eight (or more) 5ml centrifuge tubes, and add reagents as follows:

Note

Incubate at 37 ℃ for 15-30mins. Measure the absorbance at 562nm, and draw a standard curve to calculate the sample protein content. Avoid water evaporation from affecting the test results when using an incubator.

1. Cu Reagent and PBS Dilution can be stored at 2-8℃ when the reagent is not used for a long time. When the BCA reagent crystallizes and precipitates at low temperatures, it can be incubated at 37℃ to dissolve completely without affecting its use.
2. If the sample contains a lot of interfering substances, please use BCA Protein AssayKit (PC0020).
3. For your safety and health, please wear gloves and gauze mask

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