ANALYTICAL
`
`BIOCHEMISTRY
`
`81,
`
`478-480
`
`(1977)
`
`Brilliant Blue G-250
`of the Coomassie
`An Evaluation
`Dye-Binding
`Method
`for Quantitative
`Protein Determination
`
`(1) suggests that the binding of Coomassie
`A recent report of Bradford
`in an acid-denaturing
`solvent provides a
`brilliant
`blue G-250
`to protein
`rapid, reproducible,
`and sensitive method
`for quantitative
`determination
`of
`protein. Application
`of the method
`in our laboratory
`shows that it is a rapid
`and reproducible method, but that it is of limited analytical
`value since it
`shows a wide variability
`in its sensitivity
`to various proteins.
`
`MATERIALS
`
`AND METHODS
`
`from
`Coomassie brilliant blue G-250 was obtained
`Reagentprepavatiorz.
`Sigma Chemical Co. All other reagents used were of analytical grade or the
`best grade available. The protein
`reagent was prepared as described by
`Bradford
`(1).
`c (horse heart),
`cytochrome
`Protein
`solutions. Bovine serum albumin,
`(bovine pancreas), pepsinogen
`trypsin (bovine pancreas). y-chymotrypsin
`(hog stomach),
`lysozyme
`(egg white), and chymotrypsinogen
`A (bovine
`pancreas) were obtained
`from Sigma. Protein solutions were prepared
`in
`0.15 M NaCl. Protein concentrations were determined
`spectrophotometri-
`tally at 280 nm using &, values as follows: bovine serum albumin, 6.6 (3);
`y-chymotrypsin
`[an E&$, = 20.1 for Lu-chymotrypsin
`(4) was used];
`trypsin,
`15.4 (5); cytochrome C, 17.1 (6); lysozyme, 26.4 (7); pepsinogen,
`13.7 (8);
`chymotrypsinogen
`A, 20 (9).
`Protein assays. Identical
`standard protein solutions were assayed by the
`dye-binding method
`(1) and by the method of Lowry et al. (2). Average
`values of duplicate measurements
`are reported.
`
`RESULTS
`
`AND DISCUSSION
`
`assay (1)
`Figures 1 and 2 show the response curves for the dye-binding
`and the protein assay of Lowry et al. (2) as applied
`to several proteins. As
`noted in Fig. 1, bovine serum albumin and cytochrome c give results in accord
`with the results reported by Bradford
`(1). However,
`the remaining
`five pro-
`teins show a much greater variability
`than when the same proteins are assayed
`by the method of Lowry et al. (2) (Fig. 2). The data for the Lowry et al. (2)
`assay of all seven proteins describe a straight
`line with a correlation
`coefficient of 0.92. The data for the protein dye-binding
`assay of the same
`
`CopyrIght
`
`D
`
`1977
`
`by
`
`Academr
`
`Pre~5.
`
`Inc.
`
`All
`
`rights
`
`of
`
`reproductmn
`
`m
`
`any
`
`form
`
`reserved.
`
`ISSN
`
`OCW2697
`
`478
`
`GNE 2005
`Page 1
`
`

`
`SHORT
`
`COMMUNICATIONS
`
`479
`
`c
`Cyotchrome
`Chymotrypslnogen
`Pepsinogen
`
`A
`
`10 - T~YP=~
`T -Chymotryps,n
`Lysozyme
`
`9
`o
`i
`
`i?
`7
`
`the absorbance
`of
`1. Plot
`FIG.
`micrograms
`ofprotein.
`The proteins
`and Methods
`section
`for details
`of
`
`the
`versus
`assay
`the dye-binding
`in
`at 595 nm obtained
`used
`in the assay are given
`in the
`figure. See the Materials
`the measurement.
`
`is not
`line. The variability
`seven protein solutions do not fit a single straight
`due to differences
`in the absorption
`spectrum of the bound dye, since the
`absorption maximum
`of all complexes
`centered about 590-595 nm. The
`variability
`presumably
`reflects differences
`in the amount of dye bound
`since each protein except
`lysozyme gives a linear
`response of ODjss vs
`micrograms
`of protein. The application
`of this method
`to the analyses of
`conjugated proteins such as glycoproteins
`and lipoproteins must also be
`examined,
`since such proteins may also bind differential
`amounts of dye.
`The protein
`reagent was stable
`in our hands for at least 2 weeks. This
`fact, coupled with the rapidity and sensitivity of the assay, makes this assay
`useful
`for quantitative
`analysis of proteins under certain conditions.
`For
`example,
`repetitive
`analysis of a purified
`protein,
`or of
`the same
`heterogeneous
`protein solution, may be quantitated with the dye-binding
`method
`if it is first standardized
`against a more accurate method such as
`that of Lowry et al. (2). On the other hand,
`if sufficiently
`concentrated
`protein solutions are available
`(a minimum
`of 50 pgiml),
`it is our viewpoint
`that measurements
`at 280 nm can be standardized
`in a similar manner and
`can be made with more
`rapidity
`and ease. Protein
`solutions
`less
`concentrated
`than 50 pug/ml may be quantitated
`at 205 nm ( 10).
`
`GNE 2005
`Page 2
`
`

`
`480
`
`SHORT COMMUNICATIONS
`
`A
`
`Bovme serum albumtn
`Cytochrome
`c
`Chymoirypslnogen
`Pepsmogen
`Trypsm
`I -Chymotrypsln
`Lysozyme
`
`o
`x
`o
`0
`
`+
`n
`0
`
`L
`
`20
`
`40
`Jig
`
`Prate1n
`
`60
`
`80
`
`FIG. 2. Plot of the absorbance at 750 nm in the Lowry et al. (2) assay versus the micrograms
`of protein. The proteins used in the assay are given in the figure. See the Materials and
`Methods section for details of the measurement.
`
`REFERENCES
`1. Bradford, M. M. (1976) Anal. Biochem.
`72, 248-254.
`2. Lowry, 0. H., Rosebrough, N. J.. Farr. A. L., and Randall, R. J. (1951) J. Biol. Chem.
`193, 265-275.
`74, 2509-2515.
`3. Tanford, C., and Roberts, G. L., Jr. (1952) J. Amer.
`Sot.
`Chem.
`4. Egan, R., Michel, H. O., Schlueter, R.. and Jandorf. B. J. (1957)Arch. Biochern.
`Biophys.
`66, 354-365.
`5. Keil, B. (1971) in The Enzymes (P. Boyer. ed.), Vol. 3, p. 255, Academic Press, New
`York.
`55, 166- 192.
`6. Kirschenbaum, D. M. (1973) Anal.
`Biochem.
`7. Aune, K. C., and Tanford, C. (1969) Biochemistry
`8, 4579-4585.
`8. Perlmann, G. E. (1964) .I. Bio[. Chem.
`239, 3762-3766.
`9. Guy, O., Gratecos, D., Rovery. M., and Desnuelle, P. (1966)Biochim.
`404-422.
`10. Scopes, R. K. (1974) Anal.
`
`Biophys.
`
`Acta
`
`115,
`
`B&hem.
`
`59, 277-282.
`
`of Biochemistry
`Department
`State
`Unil’ersity
`Michigan
`48824
`East Lansing,
`Michigan
`Received
`Jarwar?,
`6, 1977: accepted
`
`May
`
`9, 1977
`
`JOHN PIERCE
`C.H. SUELTER
`
`GNE 2005
`Page 3