BCA Protein Assay

Materials

• Bovine serum albumin standard
• Reagent A from BCA kit
• Reagent B from BCA kit
• Cell extraction buffer as diluent
• Multichannel pipette and pipette tips
• Pipettes and pipette tips
• 96-well microplate
• Incubator set at 37°C

Procedure

1. Serial dilution for standard curve was performed using bovine serum albumin (BSA) as described below:

Vial	Volume of Diluent (µl)	Volume (µl) and source of BSA	Final BSA Concentration µg/ml
A	0	300 of Stock	2000
B	125	375 of Stock	1500
C	325	325 of Stock	1000
D	175	175 of vial B dilution	750
E	325	325 of vial C dilution	500
F	325	325 of vial E dilution	250
G	325	325 of vial F dilution	125
H	400	100 of vial G dilution	25
I	400	0	0

2. 25 µl of each standard (A-I) and each sample was added to a 96-well microplate. Samples were added in replicates of three.

3. Working reagent was prepared using a ratio of 1:50 (Reagent A:Reagent B). A green solution formed.

4. 200 µl of working reagent was added to each well using a multichannel pipette. This was performed quickly because the reaction is time sensitive.

5. The microplate was placed in an incubator for 15 minutes at 37°C. The protein in each well appeared purple.

6. Absorbance of each well was read at 595 nm using an ELISA microplate reader. Standard I was used as the blank on the reader.

7. Amount of protein in each well was calculated by plotting a standard curve on Excel.

Pictures from Procedure (click on each picture to open it in its own window)

Working reagent prior to additon to wellplates

Plotting a standard curve on Excel

Appearance of wellplates after incubation