ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 331 (2004) 255–259

www.elsevier.com/locate/yabio

Interference in the Coomassie Brilliant Blue and Pyrogallol Red protein dye-binding assays is increased by the addition

of sodium dodecyl sulfate to the dye reagents

Thomas Marshall¤ and Katherine M. Williams

Analytical Biochemistry Group, Sunderland Pharmacy School, Fleming Building, The University of Sunderland, Sunderland, SR1 3RG, UK

Received 19 January 2004Available online 19 June 2004

Abstract

We have investigated the eVect of sodium dodecyl sulfate (SDS) upon the response of the Coomassie Brilliant Blue (CBB) andPyrogallol Red-molybdate (PRM) protein dye-binding assays to interference from aminoglycosides, ampholytes, detergents, pheno-thiazines, reducing agents, and miscellaneous substances previously reported to interfere with the assays. The CBB assay was lessprone to interference than the PRM assay but gave positive interference with the detergents and the phenothiazines and negativeinterference with dextran sulfate. The PRM assay gave positive interference with the aminoglycosides, ampholytes, and phenothia-zines and negative interference with SDS, citric acid, dextran sulfate, EDTA, oxalic acid, and tartaric acid. The level of interferencevaried in the presence of diVerent proteins (albumin, gamma globulin, �1-acid glycoprotein, or lysozyme) and increased when SDSwas added to the dye reagents. 2004 Elsevier Inc. All rights reserved.

The Coomassie Brilliant Blue (CBB)1 protein dye- substances and reports, for the Wrst time, the eVect upon

binding assay [1] is widely used for protein determina-tion. The addition of sodium dodecyl sulfate (SDS) tothe dye reagent is recommended to improve the unifor-mity of response of the assay to diVerent proteins [2].The Pyrogallol Red-molybdate (PRM) assay [3,4] is analternative protein dye-binding assay, which has likewisebeen modiWed by the addition of SDS to the dye reagent[5]. The CBB assay is sensitive to interference from deter-gents [1], guanidinium hydrochloride [6], phenothiazines[7,8], polysulfates [2], reducing agents [1], and high levelsof salt [9]. The PRM assay is sensitive to interference fromaminoglycosides [3,4,8,10], organic acids [3,4], phenothia-zines [3,4,8], and high levels of amino acids [3]. The pres-ent study simultaneously compares the responses of theCBB and PRM assays to a wide range of interfering

¤ Corresponding author. Fax: +011-44-191-515-3747.E-mail address: tom.marshall@sunderland.ac.uk (T. Marshall).1 Abbreviations used: AAG, �1-acid glycoprotein; BGG, bovine

gamma globulin; BSA, bovine serum albumin; CBB, Coomassie Bril-liant Blue; L, lysozyme; PRM, Pyrogallol Red-molybdate; SDS, sodi-um dodecyl sulfate.

0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2004.04.029

interference of adding SDS to the dye reagents.

Materials and methods

Materials

The aminoglycosides gentamicin sulfate (G1914),neomycin trisulfate hydrate (N5285), paromomycin sul-fate (P9297), and tobramycin sulfate (T1783) were pur-chased from Sigma–Aldrich (Poole, Dorset, UK) andsolubilized at 10 g/L in deionized water. The gentamicinand neomycin were further diluted to 5 and 2 g/L,respectively. The ampholytes pH 2.5–4 (20% w/v), pH 4–6 (40%), pH 5–7 (40%), pH 9–11 (20%), and pH 3.5–10(40%) were purchased from Sigma and diluted to 0.5 g/L(0.05% w/v) in deionized water. The detergents SDS, Tri-ton X-100, and Tween 20 (Plus One grade, AmershamBiosciences, Chalfont St. Giles, Buckinghamshire, UK)and Nonidet P-40 (BDH Chemicals, Poole, Dorset, UK)were prepared at 5 g/L. The phenothiazines chlorproma-zine hydrochloride (C8138), Xuphenazine dihydrochloride

256 T. Marshall, K.M. Williams / Analytical Biochemistry 331 (2004) 255–259

(USP, F8548), promazine hydrochloride (P6656), thio-ridazine hydrochloride (USP, T4812), triXuoperazinedihydrochloride (T6062), and triXupromazine hydro-chloride (T2896) were purchased from Sigma and solu-bilized at 1 g/L. Fluphenazine, thioridazine, andtriXuoperazine were diluted to 0.5 g/L. The reducingagents dithiothreitol and mercaptoethanol (Plus Onegrade, Amersham Biosciences) were prepared at 100 g/L.Ammonium sulfate, cesium chloride, citric acid, EDTA,glycerol, glycine, guanidinium hydrochloride, potassiumchloride, sodium chloride, sodium nitrate, sucrose, tar-taric acid, Tris, and urea (all of analytical grade; Sigma)were prepared at 100 g/L. Dextran sulfate and oxalicacid were solubilized at 0.1 and 25 g/L, respectively.Bovine serum albumin (A7906), lysozyme (L2879), and�1-acid glycoprotein (G3643) were purchased from Sigma.Bovine gamma globulin (500-0005) was purchased fromBio-Rad Laboratories Ltd. (Hemel Hempstead, UK).The proteins were solubilized at 1 g/L and an aliquot was

mixed with an equal volume of interfering substance attwice the working concentration.

Dye reagents

The CBB reagent was freshly prepared by dissolving40 mg CBB G 250 (Serva Blue G, Serva Fine Chemicals,Heidelberg, Germany) in 40 mL of methanol, adding100 mL of 85% phosphoric acid and adjusting the volumeto 1 L with deionized water [1,2]. The CBB/SDS reagentwas prepared by adding 1 mL of 3% w/v SDS (f.c. 30mg/L) prior to Wnal adjustment of the volume to 1 L [2]. ThePRM reagent was freshly prepared by dissolving 5.9g suc-cinic acid, 0.14g sodium oxalate, and 0.5 g sodium benzo-ate in 900 mL of water and adding 40 mL of dye (60 mgPyrogallol Red (P8759; Sigma–Aldrich Ltd.) in 100 mLmethanol) and 4 mL of disodium molybdate (0.24 g/100 mL water). The reagent was adjusted to pH 2.5 with0.5 mol/L hydrochloric acid and a volume of 1 L with

Table 1Interference in the absence of protein expressed as �g BSA equivalent

The values were obtained by extrapolating the absorbance of the assay mixture (20 �L of interfering substance + 1 mL dye reagent) from the cor-responding BSA calibration curve. All absorbance values were within the linear working range of the assays. Minus values indicate absorbance val-ues 0 the reagent blank. To highlight the interference, values 60.5 �g are not shown (n D 4, %CV 0 6%). Note: 100 g/L cesium chloride, potassiumchloride, sodium chloride, sodium nitrate, and sucrose did not interfere with any of the assays and are not shown.

Substance Concentration (g/L) CBB CBB/SDS PRM PRM/SDS

Aminoglycosides Gentamicin 5 1.1 12.4 30.3Neomycin 2 1.0 12.0 28.5Paromomycin 10 4.1 10.7Tobramycin 10 1.1 11.7 27.8

Ampholytes pH 2.5–4 0.5 0.7 0.64–6 0.5 2.6 10.1 15.85–7 0.5 3.4 14.6 21.89–11 0.5 3.0 23.2 34.93.5–10 0.5 3.4 16.5 24.8

Detergents Nonidet P-40 5 1.7 30.4SDS 5 9.0 22.6Triton X-100 5 1.1 28.3Tween 20 5 6.5 17.9

Phenothiazines Chlorpromazine 1 1.1 10.3 5.3 20.5Fluphenazine 0.5 1.0 8.2 12.6 22.2Promazine 1 0.8 5.1 1.5 1.8Thioridazine 0.5 1.9 9.4 7.8 12.2TriXuoperazine 0.5 1.2 8.0 13.8 22.1TriXupromazine 1 1.0 8.8 1.3 13.2

Reducing Agents Dithiothreitol 100Mercaptoethanol 100

Miscellaneous Ammonium sulfate 100Citric acid 100 0.7 ¡2.3 ¡3.3Dextran sulfate 0.1EDTA 100 1.1 ¡1.5 ¡2.1Glycerol 100 0.6 0.8Glycine 100 1.2Guanidinium HCl 100 1.0 0.7 1.2Oxalic acid 25 1.0 ¡1.3 ¡2.1Tartaric acid 100 1.2Tris 100 0.9 1.4 0.7 0.8Urea 100 0.7 1.3

T. Marshall, K.M. Williams / Analytical Biochemistry 331 (2004) 255–259 257

deionized water [4]. The PRM/SDS reagent was pre-pared by adding 1 mL of 2.5% w/v SDS (f.c. 25 mg/L)prior to Wnal adjustment of the volume to 1 L [5].

Protein assays

Twenty microlitres of sample or BSA protein calibra-tor (0.5–2 g/L, Pierce Pre-diluted Protein Standards, Per-bio Science UK Ltd., Tattenhall, Cheshire, UK) wasmixed with 1mL of dye reagent. After 30 min, the absor-bance was measured at 595nm (CBB assay) or 600nm(PRM assay) using a Jenway 6100 Spectrophotometer(Dunmow, Essex, UK) zeroed with a water/reagent blank.

Results and discussion

Table 1 indicates the interference in the absence ofprotein based upon the assay of 20 �L of substance

mixed with 1 mL of dye reagent. The absorbance valueswere calibrated against BSA and the interferenceexpressed as �g BSA equivalent [1,4]. The CBB assaygave positive interference with the detergents, phenothi-azines, glycerol, Tris, and urea (Table 1). The addition ofSDS to the dye reagent (CBB/SDS assay) greatlyincreased the interference from the detergents (whichgave an abnormal green chromophore) and the pheno-thiazines and slightly increased the interference fromglycerol, Tris, and urea (Table 1). The SDS also resultedin low interference with the aminoglycosides, ampho-lytes, citric acid, EDTA, glycine, guanidinium hydro-chloride, oxalic acid, and tartaric acid (Table 1). ThePRM assay gave positive interference with the aminogly-cosides, ampholytes, phenothiazines, guanidiniumhydrochloride, and Tris and negative interference withcitric acid, EDTA, and oxalic acid (Table 1). The inter-ference from the ampholytes was pH dependent (Table1). The addition of SDS to the dye reagent (PRM/SDS

Table 2Interference in the presence of bovine serum albumin (BSA) expressed as �g BSA equivalent

The values were obtained by extrapolation of the absorbance of the assay mixture (10 �g BSA in 20 �L of interfering substance + 1 mL dyereagent) minus that of 10 �g BSA assayed alone. All absorbance values were within the linear working range of the assays. To highlight the interfer-ence, values 60.6 �g are not shown (n D 4, %CV 0 7%). Note: 100 g/L cesium chloride, potassium chloride, sodium chloride, sodium nitrate, andsucrose did not interfere with any of the assays and are not shown.

Substance Concentration (g/L) CBB CBB/SDS PRM PRM/SDS

Aminoglycosides Gentamicin 5 1.0 9.7 35.8Neomycin 2 9.4 34.4Paromomycin 10 4.2 13.2Tobramycin 10 1.0 9.4 33.4

Ampholytes pH 2.5–4 0.5 0.9 1.7 1.94–6 0.5 1.3 10.0 16.65–7 0.5 2.2 13.8 22.89–11 0.5 2.2 20.1 35.43.5–10 0.5 2.1 15.5 25.8

Detergents Nonidet P-40 5 9.3 27.0 2.2 3.1SDS 5 0.7 19.1 ¡7.9 ¡7.9Triton X-100 5 6.1 25.7 2.4 3.0Tween 20 5 9.8 18.9 2.2 2.2

Phenothiazines Chlorpromazine 1 1.4 8.7 4.6 18.5Fluphenazine 0.5 6.2 11.6 26.5Promazine 1 4.5 1.8 4.3Thioridazine 0.5 1.2 6.4 5.0 11.4TriXuoperazine 0.5 0.7 7.2 12.1 26.7TriXupromazine 1 1.2 6.2 3.4 15.8

Reducing Agents Dithiothreitol 100 4.2 1.1 0.7Mercaptoethanol 100 0.8

Miscellaneous Ammonium sulfate 100 ¡0.8Citric acid 100 0.7 ¡4.0 ¡11.3Dextran sulfate 0.1 ¡4.9 ¡5.1 ¡7.4 ¡7.8EDTA 100 1.5 ¡2.3 ¡6.0Glycerol 100 0.9Glycine 100 2.0 2.6 0.8Guanidinium HCl 100 1.5 2.7 2.8Oxalic acid 25 1.2 1.5 ¡3.8 ¡6.7Tartaric acid 100 0.9 1.2 ¡4.3 ¡7.3Tris 100 1.3 2.0 1.2 3.4Urea 100 0.8 0.7 1.1

258 T. Marshall, K.M. Williams / Analytical Biochemistry 331 (2004) 255–259

assay) increased the interference with all of these sub-stances (Table 1).

Table 2 indicates the interference in the presence ofBSA based upon the assay of 10�g BSA in 20�L of sub-stance mixed with 1 mL of dye reagent. In each case, theabsorbance value of the mixture minus that of 10�g BSAassayed alone was calibrated against BSA and the inter-ference expressed as �g BSA equivalent. Table 2 demon-strates interference in the presence of protein andconWrms that the addition of SDS to the dye reagentsresults in an increase in the interference. The levels ofinterference with the aminoglycosides, ampholytes, andphenothiazines (Table 2) were similar to those obtainedin the absence of protein (Table 1), the latter correspond-ing to the respective reagent blanks. However, the non-ionic detergents (Nonidet P-40, Triton X-100, and Tween20) and the reducing agents gave higher than expectedinterference with some of the assays, and SDS gave lowerthan expected interference with the CBB assay andnegative interference with the PRM and PRM/SDS

assays (Tables 1 and 2). In addition, tartaric acid gavenegative interference (and citric acid, EDTA, and oxalicacid greater than expected negative interference) with thePRM and PRM/SDS assays and dextran sulfate gavenegative interference with all four assays (Tables 1 and 2).

Table 3 indicates the levels of interference obtainedwith additional proteins using representative interferingsubstances. The values are based upon assay of 10 �g ofbovine gamma globulin (BGG), �1-acid glycoprotein(AAG), or lysozyme (L) in 20 �L of substance mixedwith 1 mL of dye reagent. In each case, the absorbancevalue of the mixture minus that of 10 �g protein assayedalone was calibrated against BSA and the interferenceexpressed as �g BSA equivalent. The results demonstratethat the patterns of interference and the eVects of SDSupon the interference are consistent with all four pro-teins but the level of interference varies from protein toprotein (Tables 2 and 3).

In conclusion, the PRM assay is more prone to inter-ference than the CBB assay but both assays show an

Table 3Interference in the presence of bovine gamma globulin (BGG), �1-acid glycoprotein (AAG), or lysozyme (L) expressed as �g BSA equivalent

The values were obtained by extrapolation of the absorbance of the assay mixture (10 �g protein in 20 �L interfering substance + 1 mL dyereagent) minus that of 10 �g protein assayed alone. All absorbance values were within the linear working range of the assays. To highlight the inter-ference, values 6 0.6 �g are not shown (n D 4, %CV 0 6%).

Substance Protein CBB CBB/SDS PRM PRM/SDS

Gentamicin (5 g/L) BGG 0.8 14.0 29.1AAG 0.9 10.6 26.0L 1.7 13.6 30.0

Ampholyte pH 3.5–10 (0.5 g/L) BGG 1.9 15.2 27.4AAG 1.8 12.9 23.7L 0.7 2.1 15.8 27.5

SDS (5 g/L) BGG 3.7 16.1 ¡2.6 ¡4.8AAG 4.7 17.9 ¡3.4 ¡5.1L 7.2 11.3 ¡5.7 ¡10.7

Triton X-100 (5 g/L) BGG 4.9 25.1 0.9AAG 4.0 24.1 0.9 ¡1.0L 4.1 21.7 1.5 ¡0.7

Chlorpromazine (1 g/L) BGG 2.1 10.4 1.7 9.9AAG 1.4 11.3 1.4 10.7L 2.7 7.1 2.8 14.0

Citric acid (100 g/L) BGG ¡1.3 ¡6.1AAG ¡2.3 ¡7.1L 0.7 ¡5.2 ¡10.3

Dextran sulphate (0.1 g/L) BGG ¡3.6 ¡4.8 ¡3.5 ¡6.0AAG ¡2.0 ¡3.2 ¡3.8 ¡5.4L ¡3.9 ¡4.4 ¡5.9

EDTA (100 g/L) BGG ¡0.9 ¡1.6AAG ¡0.9 ¡1.6L 0.7 1.9 ¡0.9 ¡0.9

Oxalic acid (25 g/L) BGG ¡1.4 ¡2.4AAG ¡1.1 ¡2.9L 1.2 ¡2.0 ¡2.6

Tartaric acid (100 g/L) BGG 0.8 1.0 ¡1.7 ¡2.6AAG 1.0 ¡1.1 ¡2.9L 0.9 2.0 ¡5.1 ¡5.1

T. Marshall, K.M. Williams / Analytical Biochemistry 331 (2004) 255–259 259

increased susceptibility to interference when SDS isadded to the dye reagents. While an appropriate reagentblank will minimize the inaccuracy resulting from theinterference of some substances (aminoglycosides,ampholytes, and phenothiazines) it will not eliminate theproblem as the level of interference changes to a varyingdegree in the presence of diVerent proteins.

References

[1] M.M. Bradford, A rapid and sensitive method for the quantitationof microgram quantities of protein utilizing the principle of pro-tein-dye binding, Anal. Biochem. 72 (1976) 248–254.

[2] M. Macart, L. Gerbaut, An improvement of the Coomassie bluedye binding method allowing an equal sensitivity to variousproteins: application to cerebrospinal Xuid, Clin. Chim. Acta 122(1982) 93–101.

[3] Y. Fujita, I. Mori, S. Kitano, Color reaction between PyrogallolRed-molybdenum (VI) complex and protein, Bunseki Kagaku 32(1983) E379–E386.

[4] N. Watanabe, S. Kamel, A. Ohkubo, M. Yamanaka, S. Ohsawa, K.Makino, K. Tokuda, Urinary protein as measured with a pyrogal-lol-red-molybdate complex manually and in a Hitachi 726 auto-mated analyzer, Clin. Chem. 32 (1986) 1551–1554.

[5] J.L. Orsonneau, P. Douet, C. Massoubre, P. Lustenberger, S. Ber-nard, An improved Pyrogallol Red-molybdate method for deter-mining total urinary protein, Clin. Chem. 35 (1989) 2233–2235.

[6] J.C. Bearden, Quantitation of submicrogram quantities of proteinby an improved protein-dye binding assay, Biochim. Biophys.Acta 533 (1978) 525–529.

[7] T. Marshall, K.M. Williams, Drug interference in the Bradfordand 2,2’-bicinchoninic acid protein assays, Anal. Biochem. 198(1991) 352–354.

[8] K.M. Williams, S.J. Arthur, G. Burrell, F. Kelly, D.W. Phillips,T. Marshall, An evaluation of protein assays for quantitativedetermination of drugs, J. Biochem. Biophys. Methods 57(2003) 45–55.

[9] T.A. Franklin, G.W. Malaney, R.D. Tanner, Deviations in thestandard curve used in the Bradford protein analysis: eVect ofhigh salt levels, J. Microb. Biotechnol. 1 (1986) 78–86.

[10] T. Marshall, K.M. Williams, Antibiotic interference in proteinassays: comparative evaluation of six photometric methods, Anal.Biochem. 322 (2003) 275–278.