Michael Court
Updated March 2003
Protein assay by BCA (Pierce) method
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Make up series of protein standard (bovine serum albumin) concentrations in 2 ml
polypropylene tubes (do in duplicate):
Final conc
200 ug/mL
400 ug/mL
Volume of albumin (2 mg/mL)
5 uL
10 uL
Volume of diluent (water)
45 uL
40 uL
600 ug/mL
800 ug/mL
1000 ug/mL
1200 ug/mL
15 uL
20 uL
25 uL
30 uL
35 uL
30 uL
25 uL
20 uL
Add 50 uL unknown protein samples to 2 ml polypropylene tubes.
a. Do in duplicate.
b. If protein concentration likely to be > 1 mg/mL then dilute sample appropriately in
water.
c. Suggest using serial dilution – ½; ¼; 1/8; 1/16.
Make up sufficient solution A and B.
a. Mix 1 part solution B with 50 parts solution A.
b. Count up the number of tubes (remember to count standards and duplicates).
c. You will need at least this many mL of solution A+B.
d. E.g. 6x2 standards + 10 samples x 2 dilutions x 2 duplicates = 52 mLs – suggest
making up > 60 mL
Add 1 mL solution A/B to each tube and mix
Incubate at 370C for 20-30 minutes (or can let stand at room temperature for 2 hours)
Read absorbance at 562 nM in spectrophotometer using 1 mL plastic cuvettes (only need
to use 1 cuvette – wash between samples)
a. Don’t allow time to elapse between readings since reaction continues albeit slowly at
room temperature.
Use standard curve function of spectrophotometer (linear regression) to calculate protein
concentrations (or can use raw data and Excel spreadsheet)