- Mary Johnson Ph. D.mary at labome dot comSynatom Research, Princeton, New Jersey, United States
Accurate protein quantitation is essential to all experiments related to proteins in a multitude of research topics in molecular biology, cell biology, biochemistry, developmental biology, and neuroscience.
Different methods have been developed to quantitate proteins in a given assay for the past century, for the total protein content and for a single protein.
Total protein quantitation methods comprise traditional methods such as the measurement of UV absorbance at 280 nm, Bicinchoninic acid (BCA) and Bradford assays, as well as alternative methods like Lowry or novel assays developed by commercial suppliers. Typically commercial suppliers provide a well-designed, convinient kit for each type of the assays.
Individual protein quantitation methods include enzyme-linked immunosorbent (ELISA) assay, western blot analysis, and more recently, mass spectrometry, among others.
Here we discuss some of the common methods used to determine protein concentration, highlighting their utilities and limitations. It is important to note that none of these protein assays is either specific to proteins, which means that non-protein components may interfere, or uniformly accurate and compatible to all proteins.
Regulations or laws from government agencies or trade groups may require specific methods for the determination of total proteins. For example, International Serum Industry Association (www.serumindustry.org), a trade group for serum providers, requires the Biuret method for the determination of protein contents in serum products.
Table I summarizes the common total protein quantitation assays. It is important for the researchers to evaluate the compatibility of each assay with their sample types, assay range, sample volume , and the availability of a suitable spectrophotometer, as well as the time and cost to perform each assay.
|UV absorption||280 nm||tyrosine and tryptophan absorption||0.1-100 ug/ml||small sample volume, rapid, low cost||incompatible with detergents and denaturating agents, high variability|
|Bicinchoninic acid||562 nm||copper reduction (Cu2+ to Cu1+), BCA reaction with Cu1+||20-2000 ug/ml||compatible with detergents and denaturating agents, low variability||low or no compatibility with reducing agents|
|Bradford or Coomassie brilliant blue||470 nm||complex formation between Coomassie brilliant blue dye and proteins||20-2000 ug/ml||compatible with reducing agents, rapid||incompatible with detergents|
|Lowry||750 nm||copper reduction by proteins, Folin-Ciocalteu reduction by the copper-protein complex||10-1000 ug/ml||high sensitivity and precision||incompatible with detergents and reducing agents, long procedure|
Aromatic amino acids tyrosine and tryptophan give proteins the characteristic ultraviolet (UV) absorption spectrum at 280 nm, which is routinely used to estimate protein concentration. Phenylalanine and disulfide bonds also contribute the absorption at that wavelength, albeit slightly. This method is simple, and requires an extremely small sample volume since new spectrophotometers employ a sample retention system during the measurement. However, the protein sample must be pure and does not contain any non-protein components with the same absorption spectrum, such as the contamination of nucleic acids . This method is quickest, but error-prone.
The bicinchoninic acid (BCA) assay was invented by Paul K. Smith in 1985 at Pierce Chemical Company, the major distributor of this assay [Thermo Scientific Pierce Protein Assay Technical Handbook http://www.piercenet.com/files/1602063_PAssayHB_122910.pdf]. Both BCA assay and Lowry assay are based on the conversion of Cu2+ to Cu1+ under alkaline conditions. This conversion is defined as Biuret reaction. This reaction is influenced by four amino acid residues (cysteine, cystine, tyrosine, and tryptophan) and also by the peptide backbone.
BCA is a specific chromogenic reagent for Cu1+ and in the second step of the reaction two BCA molecules react with one Cu1+ ion. The amount of Cu2+ reduced is a function of protein concentration that can be determined spectrophotometrically by a color change of the sample solution into purple, which absorbs at 562 nm. The absorbance is directly proportional to the amount of protein present in the solution and it can be estimated by comparison with a known protein standard, such as bovine serum albumin (BSA)   .
BCA assay is more tolerant to a range of ionic and nonionic detergents such as NP-40, Triton X-100 and denaturating agents like urea and guanidinium chloride, that tend to interfere with other colorimetric protein assay such like Lowry (Table 2) . Some chemical molecules, for instance, reducing sugars, can interfere with BCA assay. The effects of these interferences can be eliminated or reduced through several strategies such as removing the interfering substances through dialysis, gel filtration or if the protein concentration is high enough, by diluting the sample.
|Emulgen||Glycine, pH 2.8|
|Guanidine HCl||Ammonium Sulfate|
|Sodium Acetate, pH 5.5||SDS|
Thermo Fisher Pierce has recently introduced a new version of the classical Pierce BCA Protein assay compatible with reducing agents such as dithiothreitol (DTT), 2-merceptoethanol (BME), TCEP and other disulfide reducing agents [http://www.piercenet.com/files/1602063_PAssayHB_122910.pdf].
Compared to other methods BCA assay is one of the most sensitive (it can detect proteins at concentrations as low as 5 ug/mL), it has less variability than others (i.e., Bradford assay), and it can be used to measure a wide range of protein concentration.
Bradford assay, originally described by Dr. Marion Bradford in 1976, is one of the popular methods to determine protein concentration. It relies on the formation of a complex between Coomassie brilliant blue G-250 dye and proteins in solution. The free dye exists in four different ionic forms. The more anionic blue form binds to proteins and has an absorbance at 590 nm. The protein concentration can be evaluated by determining the amount of dye in the blue ionic form and by measuring the absorbance of the solution at 595 nm using a spectrophotometer . The dye binds mostly to arginine, tryptophan, tyrosine, histidine, and phenylalanine residues of the protein .
Most researchers use BSA as the protein standard since it is inexpensive and easily available but it is not always suitable. Indeed, one disadvantage of using BSA is that it exhibits a strong dye response and may under-measure the protein content. Immunoglobulin G (IgG) or lysozyme, or other protein standard should be used depending on the sample protein type .
One advantage of this method is its compatibility with reducing agents used to stabilize proteins in solution, which are not compatible with Lowry assay and in some extent with the BCA assay. The limitation of Bradford assay is the incompatibility with low concentration of detergents, which are routinely used to solubilize membrane proteins. However, the detergent substances can be removed by gel filtration, dialysis, or precipitation of the proteins with calcium phosphate . A further advantage is the possibility to measure high molecular weight (MW) proteins since the dye does not bind to peptides with low MW  . However, very low level of non-ionic detergent, such as Triton X-100 at a final concentration of 0.008% (v/v), may improve sensitivity and variability of the Bradford assay .
Lowry assay, proposed by Oliver H. Lowry in 1951, is based on two chemical reactions. The first reaction isthe reduction of copper ions under alkaline conditions, which forms a complex with peptide bonds (Biuret reaction). The second is the reduction of Folin-Ciocalteu reagent by the copper-peptide bond complex, which subsequently causes a color change of the solution into blue with an absorption in the range of 650 to 750 nm detectable with a spectrophotometer . The amount of protein in the sample can be estimated using a standard curve of a selected standard protein solution such as BSA.
The advantages of this assay are its sensitivity, and most importantly, accuracy. However, it requires more time than other assays and many compounds commonly used in buffers for protein preparation (such as, detergents, carbohydrates, glycerol, Tricine, EDTA, Tris) interfere with Lowry assay and form precipitates (Table 3) . Nonetheless, the effect of these substances can be reduced by diluting out the sample but only if the protein concentration is sufficiently high . Moreover, it has been shown that the time to perform this assay can be reduced through raising temperatures or using a microwave oven .
|Potassium compounds||Sulfhdryl compounds|
A crucial step of many experiments is the quantitation of a specific protein in solution. Several techniques have been employed to accomplish that. The common ones are ELISA, western blot analysis, and mass spectrometry. ELISA and Western blot are discussed in Antibody Applications, mass spec is discussed here briefly.
Protein mass spectrometry is an emerging method for protein quantitation. Besides protein characterization an important step in proteomic analysis is the possibility to quantify a specific protein. Many techniques have been introduced and implemented for protein quantitation by mass spectrometry. Normally, stable heavier isotopes of carbon (13C) or nitrogen (15N) are added to one sample (peptides or proteins) while corresponding light isotopes (12C and 14N) are added to a second sample (internal standard) which are mixed in the analysis. Due to mass difference it is possible to analyze the ratio of the two samples peak intensities by a mass analyzer which corresponds to their relative abundance ratio. A second method to quantify proteins by mass spectrometry can be done without labeling the samples (i.e., with matrix assisted laser desorption/ionization - MALDI analysis). Here, we want to highlight the possibility to use a universal approach as mass spectrometry to perform both quantitative and qualitative assays in one measurement. Nonetheless this method requires instrumentations that any laboratory may not afford and this may limit the utility of this approach  .
Labome surveyed over 100 randomly selected peer-reviewed publications citing total protein assays. Thermo Fisher Pierce and Bio-Rad Laboratories are identified as the major suppliers of total protein assay kits (Table 4). The most commonly used methods are BCA and Bradford.
|Thermo Fisher Pierce||63|
|Thermo Fisher Pierce||11|
Almost all BCA protein assays cited in the surveyed cohort were provided by Pierce, where one of the scientists invented the assay many years ago. Thermo Fisher purchased Pierce a number of years ago. Thermo Fisher Pierce BCA assays were used to study thyroid carcinogenesis , the role of VLDLR in cell division , S100A4 posttranslational modifications , hnRNP H and hnRNP F proteins as silencers of fibroblast growth factor receptor 2 , the mechanism underlying increased StAR and aromatase activities in endometriosis , the effect of heparanase on cell adhesion and spreading , the regulation and the role of NAG-1 expression in VES-treated PC-3 human prostate carcinoma cells , the role of IGF-1R in photoreceptor function , the role for Dlx5 in osteoblast-osteoclast coupling , and many others , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and . Its Micro BCA protein assay kits were used to study immunocytochemical localization of osteopontin in avian eggshell gland and eggshell , to study the recovery following controlled cortical impact (CCI) injury in rats , and the membrane localization of hepatitis C virus core protein and viral propagation .
Sigma BCA reagent  and Bio-Rad BCA protein assay kit were cited as well.
Thermo Fisher Pierce is also one of major suppliers of Bradford assay kits. Pierce Coomassie/Bradford assay kits were used to evaluate expression of chapeone protein ERp29 , the role for YKL-40 in modulating the biological activity of basic fibroblast growth factor , among many other research topics, , , , , , , , , and .
Bio-Rad Bradford protein assay reagents were cited in a numbe of publications , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,  and .
The best standard for protein quantitation is the same protein as the one being examined. However, this is unlikely under most circumstances. BSA (bovine serum albumin) is the most commonly used protein standard. It has limitations. It is more sensitive in Bradford essay than other proteins, thus the concentration of the protein sample is likely underestimated . In addition, BSA, as a serum protein, can not be prepared/obtained at a very high purity, since it binds to many other factors. Other proteins, such as immunoglobulin G, and lysozyme have been used as protein standards as well.
No. It is very important to ascertain that the sample datapoint fall within the standard curve ranges. The dilution of either or both sample and BSA should be adjusted.
- Olson B, Markwell J. Assays for determination of protein concentration. Curr Protoc Protein Sci. 2007;0:Unit 3.4 PMID 18429326
- Smith P, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76-85 PMID 3843705
- Walker J. The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol. 1994;32:5-8 PMID 7951748
- Kruger N. The Bradford method for protein quantitation. Methods Mol Biol. 1994;32:9-15 PMID 7951753
- Friedenauer S, Berlet H. Sensitivity and variability of the Bradford protein assay in the presence of detergents. Anal Biochem. 1989;178:263-8 PMID 2473666
- Waterborg J, Matthews H. The lowry method for protein quantitation. Methods Mol Biol. 1984;1:1-3 PMID 20512668
- Oganesian A, Armstrong L, Migliorini M, Strickland D, Bornstein P. Thrombospondins use the VLDL receptor and a nonapoptotic pathway to inhibit cell division in microvascular endothelial cells. Mol Biol Cell. 2008;19:563-71 PMID 18032585
- Dianzani C, Brucato L, Gallicchio M, Rosa A, Collino M, Fantozzi R. Celecoxib modulates adhesion of HT29 colon cancer cells to vascular endothelial cells by inhibiting ICAM-1 and VCAM-1 expression. Br J Pharmacol. 2008;153:1153-61 PMID 18084316
- Petersen A, Carlsson T, Karlsson J, Jonhede S, Zetterberg M. Effects of dexamethasone on human lens epithelial cells in culture. Mol Vis. 2008;14:1344-52 PMID 18648526
- Bonilha V, Rayborn M, Shadrach K, Li Y, Lundwall A, Malm J, et al. Semenogelins in the human retina: Differences in distribution and content between AMD and normal donor tissues. Exp Eye Res. 2008;86:150-6 PMID 18036592
- Covington H, Tropea T, Rajadhyaksha A, Kosofsky B, Miczek K. NMDA receptors in the rat VTA: a critical site for social stress to intensify cocaine taking. Psychopharmacology (Berl). 2008;197:203-16 PMID 18097654
- Rauh A, Windischhofer W, Kovacevic A, DeVaney T, Huber E, Semlitsch M, et al. Endothelin (ET)-1 and ET-3 promote expression of c-fos and c-jun in human choriocarcinoma via ET(B) receptor-mediated G(i)- and G(q)-pathways and MAP kinase activation. Br J Pharmacol. 2008;154:13-24 PMID 18362896 CrossRef
- Fan H, Zhao Z, Cheng J, Su X, Wu Q, Shan Y. Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma. World J Gastroenterol. 2009;15:2020-6 PMID 19399937
- Peng T, Chen J, Mao W, Liu X, Tao Y, Chen L, et al. Potential therapeutic significance of increased expression of aryl hydrocarbon receptor in human gastric cancer. World J Gastroenterol. 2009;15:1719-29 PMID 19360915
- Maggio-Price L, Treuting P, Bielefeldt-Ohmann H, Seamons A, Drivdahl R, Zeng W, et al. Bacterial infection of Smad3/Rag2 double-null mice with transforming growth factor-beta dysregulation as a model for studying inflammation-associated colon cancer. Am J Pathol. 2009;174:317-29 PMID 19119184 CrossRef
- Xu L, Chen Y, Song Q, Xu D, Wang Y, Ma D. PDCD5 interacts with Tip60 and functions as a cooperator in acetyltransferase activity and DNA damage-induced apoptosis. Neoplasia. 2009;11:345-54 PMID 19308289
- Zhou H, Yang H, Li Y, Wang Y, Yan L, Guo X, et al. Changes in Glial cell line-derived neurotrophic factor expression in the rostral and caudal stumps of the transected adult rat spinal cord. Neurochem Res. 2008;33:927-37 PMID 18095158
- Yeh J, Kim B, Gaines L, Peresie J, Page C, Arroyo A. The expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation. Reprod Biol Endocrinol. 2008;6:35 PMID 18710573 CrossRef
- Sasaki T, Nakamura T, Rebhun R, Cheng H, Hale K, Tsan R, et al. Modification of the primary tumor microenvironment by transforming growth factor alpha-epidermal growth factor receptor signaling promotes metastasis in an orthotopic colon cancer model. Am J Pathol. 2008;173:205-16 PMID 18583324 CrossRef
- Salvi A, Bongarzone I, Miccichè F, Arici B, Barlati S, De Petro G. Proteomic identification of LASP-1 down-regulation after RNAi urokinase silencing in human hepatocellular carcinoma cells. Neoplasia. 2009;11:207-19 PMID 19177205
- Halappanavar S, Stampfli M, Berndt-Weis L, Williams A, Douglas G, Yauk C. Toxicogenomic analysis of mainstream tobacco smoke-exposed mice reveals repression of plasminogen activator inhibitor-1 gene in heart. Inhal Toxicol. 2009;21:78-85 PMID 18925475
- Ralhan R, DeSouza L, Matta A, Chandra Tripathi S, Ghanny S, Datta Gupta S, et al. Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ labeling, multidimensional liquid chromatography, and tandem mass spectrometry. Mol Cell Proteomics. 2008;7:1162-73 PMID 18339795 CrossRef
- Leemput J, Masson C, Bigot K, Errachid A, Dansault A, Provost A, et al. ATM localization and gene expression in the adult mouse eye. Mol Vis. 2009;15:393-416 PMID 19234633
- Stoscheck C. Quantitation of protein. Methods Enzymol. 1990;182:50-68 PMID 2314256