Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

BCA-based Protein Assays
BCA Protein Assay – Reducing Agent Compatible
The Thermo Scientific Pierce BCA Assay is always compatible
with more detergents, buffers/salts and solvents than any other
colorimetric protein assay. Now it’s compatible with reducing
agents at concentrations routinely used in protein sample buffers!
The BCA Assay provides one of the most accurate measurements
of protein concentration in biological samples available.
Although the BCA Assay is compatible with more detergents,
buffers/salts and solvents than any colorimetric protein assay,
the presence of disulfide reducing agents, including dithiothreitol
(DTT) and 2-mercaptoethanol interferes with the assay. The BCA
Protein Assay Kit – Reducing Agent Compatible (Product # 23250)
provides all the advantages of the original BCA Assay as well as
compatibility with reducing agents at concentrations routinely
used in protein sample buffers (Figures 1 and 2).
Highlights:
• Compatible with up to 5mM DTT, 35mM 2-mercaptoethanol or
10mM TCEP
• No protein precipitation required
• Linear working range: 125-2,000µg/mL
• Sample volume: 25µL
• Compatible with most ionic and nonionic detergents
• Significantly less protein:protein variation than coomassie
(Bradford)-based methods
• Colorimetric method; measure at 562nm
• Easy-to-use protocol (Figure 2)
Reference
Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal.
Biochem. 150, 76-85.
25µL sample
+25µL Compatibility Reagent
in Reconstitution Buffer
Eliminate reducing
agent interference
Incubate
15 minutes at 37°C
Add 1mL BCA
Working Reagent
Perform Assay
Incubate
30 minutes at 37°C
Cool sample 5-10
minutes at room temperature
Read at 562nm
Spectrophotometer
Figure 2. Thermo Scientific Pierce BCA Protein Assay – Reducing Agent
Compatible protocol.
Net Absorbance (562nm)
Net Absorbance (562nm)
Net Absorbance (562nm)
1.2
1.0
0.8
0.6
1.2
1.0
0.8
0.6
0.4
0.2
0
1.2
1.0
0.8
0.6
0.4
0.2
– 2ME
+ 2ME
0
0 500 1,000 1,500 2,000
BSA Concentration (µg/mL)
Figure 1. Thermo Scientific Pierce BCA Protein Assay – Reducing Agent
Compatible produces a linear standard curve in the presence of reducing
agents. Color response curves for BSA after treatment with Reducing Agent
Compatible Reagent in the presence and absence of 5mM DTT, 35mM
b-mercaptoethanol and 10mM TCEP.
Ordering Information
5mM DDT
0.4
– DDT
0.2
+ DDT
0
0 500 1,000 1,500 2,000
BSA Concentration (µg/mL)
10mM TCEP
0 500 1,000 1,500 2,000
BSA Concentration (µg/mL)
35mM β-Mercaptoethanol
Product Description Pkg. Size
23250 BCA Protein Assay Kit –
Reducing Agent Compatible
Sufficient reagents to perform 250 standard
tube assays.
Includes: BCA Reagent A
BCA Reagent B
Compatibility Reagent
Reconstitution Buffer
Albumin Standard (2mg/mL)
750mL
250mL
25mL
10 x 20mg
15mL
10 x 1mL
ampules
23252 Microplate BCA Protein Assay Kit –
Reducing Agent Compatible
Sufficient reagents for 1,000 microplate assays.
Includes: BCA Reagent A
BCA Reagent B
Compatibility Reagent
Reconstitution Buffer
Albumin Standard (2mg/mL)
96-Well Microplates
– TCEP
+ TCEP
Kit
250mL
25mL
48 x 10mg
15mL
10 x 1mL
ampules
20/pkg.
18
For more information, or to download product instructions, visit www.thermoscientific.com/pierce