Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

Pierce 660nm Protein Assay
Methods:
Spectral Analysis: The absorption spectra from 340 to 800nm
were recorded using a Varian Cary Spectrophotometer of the
following component combinations: the Pierce 660nm Protein
Assay Reagent alone and in the presence of the transition metal;
100µg of bovine serum albumin (BSA) and reagent with and
without metal; and 200µg of BSA with the reagent and metal.
Absorbance (660nm)
1.5
1.0
0.5
BSA
BGG
Typical Response Curves
Test Tube Procedure: To each test tube containing 0.1mL of BSA
or BGG standard replicate (25, 50, 125, 250, 500, 750, 1000, 1500
and 2000µg/mL) in saline, 1.5mL of the Pierce 660nm Protein
Assay Reagent was added, mixed well and incubated at room
temperature for 5 minutes. The absorbance of all samples and
controls was measured at 660nm. The average absorbance for
the blank replicates (control) was subtracted from the absorbance
for individual standard replicates. A standard curve was
generated by plotting the average blank-corrected 660nm
measurement for each standard versus its concentration. For
a comparison study, the standard Bio-Rad Bradford Assay was
performed as per manufacturer’s directions.
Net Absorbance (660nm)
3
2
1
BSA
BGG
0
0 500 1,000 1,500 2,000 2,500
Protein (µg/mL)
Figure 2. Typical color response curves using the test tube procedure. The
linear detection ranges are 25-2,000µg/mL for bovine serum albumin (BSA) and
50-2,000µg/mL for bovine gamma globulin (BGG). The average absorbance for
the blank replicates (control) was subtracted from the absorbance for
individual standard replicates.
Microplate Procedure: To each well containing 0.01mL of BSA or
BGG standard replicate (25, 50, 125, 250, 500, 750, 1000, 1500 and
2000µg/mL) in saline, 0.15mL of the Pierce 660nm Protein Assay
Reagent was added. The plate was covered with sealing tape,
mixed for one minute on a plate shaker and incubated at room
temperature for five minutes. The plate reader was set to 660nm
and using the control as a blank, the absorbance of all samples
was measured. A standard curve was generated by plotting the
average blank-corrected 660nm measurement for each standard
versus its concentration.
0
0 500 1,000 1,500 2,000 2,500
Protein (µg/mL)
Figure 3. Typical color response curves using the microplate procedure. The
linear detection range is 50-2,000µg/mL for bovine serum albumin (BSA) and
bovine gamma globulin (BGG). The average absorbance for the blank replicates
(control) was subtracted from the absorbance for individual standard replicates.
Net Absorbance (660nm)
3
2
1
Thermo Scientific Pierce
660 Protein Assay
Bio-Rad Protein Assay
0
0 500 1,000 1,500 2,000 2,500
BSA (µg/mL)
Figure 4. Performance comparison of Bradford Protein Assay versus the
Thermo Scientific Pierce 660nm Protein Assay. Assays were performed
according to the standard test-tube procedure using 100µL of BSA. The Pierce
660nm Protein Assay has a greater linear range of 25-2,000µg/mL, compared
with the Bradford Assay, which has a linear range of only 125-1,000µg/mL.
References
1. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.
72, 248-254.
2. Gornall, A.G. (1949). Determination of serum proteins by means of the biuret reaction.
J. Biol. Chem. 177, 751-766.
3. Lowry, O.H. (1951). Protein measurement with Folin-Phenol reagent. J. Biol. Chem.
193, 265-275.
4. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid.
Anal. Biochem. 150, 76-85.
5. Stoscheck, C.M. (1987). Protein assay sensitive at nanogram levels. Anal. Biochem.
160, 301-305.
Ordering Information
Product Description Pkg. Size
22660 Pierce 660nm Protein Assay Reagent 750mL
Sufficient reagent for 500 standard assays and
5,000 microplate assays.
22662 Pierce 660nm Protein Assay Kit
Sufficient reagent to perform 300 standard assays
and 3,000 microplate assays.
Contains: Pierce 660nm Protein Assay Reagent
Kit
450mL
Pre-Diluted Protein Assay Standards,
Bovine Serum Albumin (BSA) Set
3.5mL each of 125- 2,000mg/mL BSA
22663 Ionic Detergent Compatibility Reagent
Sufficient for treating 100mL Pierce 660nm
Protein Assay Reagent.
5 pouches, 1 gram each
5 x 1g
14
For more information, or to download product instructions, visit www.thermoscientific.com/pierce