BCA Assay for Total Protein (Range 5-500 µg/mL)
A) Prepare Stock 0.5 mg/mL BSA Solution (kit comes with 2mg/mL BSA standard)
B) Prepare 15 mL BCA Reagent (Combine 15 mL Kit RgtA with 300 µL Kit Rgt B in
clean glass or plastic container)
C) Prepare Samples and complete assay as indicated:
Note: For the BCA assay it is important to remove DTT, imidazole, EDTA and other
reagents that may interfere with color generation (see Pierce manual)
Estimate the concentration of your unknown sample by A280 absorbance (if practical)
using an approximate molar extinction coefficient of 1mg/mL per 280 nm absorbance
unit. Dilute sample based on this estimate so that it falls in the 5-500 µg./mL
concentration range of the assay.
Prepare the standards shown in the table below.
Standard
Blank
1
2
3
4
5
6
7
[BSA] (µg/mL)
0
20
50
100
200
300
400
500
Stock BSA (µL)
0
2
5
10
20
30
40
50
MQ H2O (µL)
50
48
45
40
30
20
10
0
D) Combine 50 µL of each sample and standard to be assayed with 1 mL of BCA
reagent, vortex briefly.
E) Incubate at 37 C for 30 minutes (Use cell incubator)
F) Transfer 250 µL of each sample and standard to a clean polystyrene µ-Plate and read
the absorbance at 562 nm.