Spectrophotometric quantitation of anchorage-dependent cell numbers using extraction of naphthol blue-black-stained cellular protein

A convenient method is described by which the actual or relative number of cells in anchorage culture is determined. After removal of the growth medium, cells are subjected to a double-fixation procedure. The cellular protein content is subsequently quantitatively stained with naphthol blue-black. After a period of removal of unbound stain, dye-protein complexes are hydrolytically released and measured spectrophotometrically at 620 nm. A linear correlation exists (r = 0.994) between cell concentration, in the range 3 X 10(4) to 8 X 10(5) cells/ml of final assay volume, and absorbance up to reading values of 3.8. The technical reproducibility of the assay, as judged from assessments of cell numbers in suspension culture, displays a coefficient of variation of 5%. The method was developed for 9.6-cm2 culture dishes, but it should be possible to transform it for the use of microtiter plates.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H₄PteGlu_n has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD–serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD–serine is quantified by comparison with standards. This method was used to determine the K_m and k_cat values for 5,10-CH₂–H₄PteGlu₅ of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH₂–H₄PteGlu₅ for an SHMT from any organism.     

Measurement of protein using bicinchoninic acid

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu²⁺ in the BCA Kit’s reagent B (4% cupric sulfate) in a manner similar to that of the protein.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Measurement of protein using flow injection analysis with bicinchoninic acid

We have used the bicinchoninic acid reagent developed by Pierce Chemical Co. to measure proteins in a simple flow injection analyzer. The sensitivity is comparable to that of the Lowry method and no pipetting of reagents is needed. Results are obtained in less than 1 min and samples may be run at a rate of 60/h. The response is linear over a range of protein concentration (0-10 micrograms) and sample size (5-20 microliters) convenient for most analytical requirements. A peristaltic pump, a controlled-temperature water bath, and a spectrophotometer with flow cuvette are the only special apparatus required.     

Interference by lipids in the determination of protein using bicinchoninic acid

Bicinchoninic acid forms the basis of an analytical method for the determination of protein. The reagent produces a purple complex with cuprous ion (Cu+) in an alkaline environment and is the basis for the monitoring of cuprous ions produced in the reactions of proteins with alkaline Cu2+. This method of protein determination was reported to have greater tolerance to many commonly encountered interfering compounds, when compared to the Lowry technique. However, we have found the bicinchoninic acid technique to produce erroneously high values for protein when common membrane phospholipids were included in the assay. Phospholipids in the presence of bicinchoninic acid produced an absorbance peak similar to that produced by protein. This absorbance was linear with concentration, however, the slope varied for individual phospholipids. The combined absorption of phospholipid and protein was not strictly additive. The results indicate that the presence of appreciable quantities of lipid in samples can cause significant error in the analysis of protein by the bicinchoninic acid procedure.     

pH and buffering in the bicinchoninic acid (4,4'-dicarboxy-2,2'-biquinoline) protein assay

The HCO3/CO3(2-) buffer used in the bicinchoninic acid (BCA) protein assay has only weak buffering capacity at the recommended pH (11.25). Consequently the assay is rather sensitive to interference from effectively acid or alkaline samples, particularly in the micro method. Adjustment of pH in these alkaline solutions of high [Na+] is complicated by Na+ errors on the pH electrode. Hence it is recommended to prepare the buffers from known amounts of NaHCO3 and Na2CO3, and to reduce the pH to around 10.7; this offers much better buffering capacity with only a limited reduction in color development.     

Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation

The colored complex formed between Cu+ and bicinchoninic acid is the basis of the bicinchoninic acid protein assay (P. K. Smith, R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B.J. Olson, and D.C. Klenk (1985) Anal. Biochem. 150, 76-85). Studies show that cysteine, tryptophan, tyrosine, and the peptide bond are capable of reducing Cu2+ to Cu+. Electrochemical studies and the magnitude of the color changes observed when the reaction is carried out at 37 degrees C indicate that tryptophan, tyrosine, and the peptide bond are not completely oxidized at this temperature. When the reaction temperature is increased to 60 degrees C, significantly more color formation is observed for these three groups. Studies with di-, tri-, and tetrapeptides and with proteins indicate that the extent of color formation is not the sum of the contributions of the individual color producing functional groups. Compounds with functional groups similar to those of cysteine, cystine, tyrosine, or tryptophan are shown to react with the bicinchoninic acid reagent. The color formed by these compounds in the presence of bovine serum albumin cannot be compensated for by using a reagent blank containing an identical concentration of the interfering compound.     

Quantification of protein in dilute and complex samples: modification of the bicinchoninic acid assay

The colorimetric assay using bicinchoninic acid (BCA) as test reagent is useful for quantitative protein determinations due to its high sensitivity, ease, and tolerance to various contaminations present in biological samples or added during purification. For removal of interfering substances, protein precipitations have been described. Yet, obstructions became apparent with diluted and complex samples. Therefore we tested different solvents for removal of such interfering contaminants from the protein precipitate, and 1 M HCl was identified as the useful washing agent. The protocol described allows simple and accurate microdetermination in microtiter plates of proteins from complex samples.     

Determination of protein covalently bound to agarose supports using bicinchoninic acid

A method using bicinchoninic acid (BCA) for the direct determination of protein covalently bound to agarose is described. The method involves the preparation of a standard curve using solubilized protein, the determination of the slurry concentration of the gel sample, the colorimetric assay of the gel slurry using BCA in a serum separator device, and the calculation of the amount of protein bound to the gel using the standard curve of the solubilized protein. The use of the BCA method for direct estimation of immobilized protein agrees well with the "balance" method which depends upon the measurement of protein depletion and yields highly reproducible results with a variety of coupling chemistries commonly used with agarose beads. The use of commercially available serum filters provides for a fast, less than 1 h, and convenient analytical format.     

Modification of the bicinchoninic acid protein assay to eliminate lipid interference in determining lipoprotein protein content.

The bicinchoninic acid (BCA) assay method for the determination of protein has been investigated for its utility in measuring the protein content of plasma lipoproteins. Although other methods, principally those based on the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275) have been extensively used for this purpose, the tolerance of the BCA method to many commonly encountered detergents and buffers offers a definite advantage over the Lowry-based methods. In this study, lipoprotein protein values obtained by the BCA method were compared to a standard modification of the Lowry et al. procedure since this assay forms the basis of much of the relevant literature. The standard BCA assay was found to overestimate the protein content of very low density lipoprotein by approximately 70% and low density lipoprotein by approximately 30%; high density lipoprotein values compared favorably. Overestimations by the BCA assay paralleled the relative phospholipid content of the lipoprotein fractions. This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis. In the presence of this detergent, BCA assay measurements for these three lipoprotein fractions were 97, 90, and 98%, respectively, of the reference assay values.     

Quantitative benedict test using bicinchoninic acid

Cupric ion (Cu2+), in complex form, functions as a selective oxidizing agent for a variety of compounds in the qualitative Benedict test. Cupric ion is reduced to cuprous ion (Cu+) in the reaction. We found that the cuprous ion formed in this type of redox reaction could be detected and quantified using 2,2'-bicinchoninic acid. This reagent produced an intense purple complex with cuprous ion. The color development in this modified Benedict test was dependent on pH, temperature, and time. The reaction is insensitive to ethanol and sodium dodecyl sulfate. This improved method of the Benedict test has enhanced sensitivity and makes the quantitation of compounds possible. This method should be useful for studies of Benedict-positive compounds which are available only in small amounts.     

Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions

The bincinchoninic acid protein assay has been adapted for use with microtiter plates and a plate reader to reduce the time needed for analyses. The total volume of the assay was reduced to 0.21 ml with various ratios of sample to reagent volume being used. The assay is sensitive to a limit of less than 1 microgram of bovine serum albumin. The assay is insensitive to the presence of the detergents sodium dodecyl sulfate, Triton X-100, and deoxycholate. In addition, the method has been adapted for determining the protein concentrations of sucrose density gradient fractions, which eliminates the need for removing the sucrose prior to assay.     

Estimation of peptide concentration by a modified bicinchoninic acid assay

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 °C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 °C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.     

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu²⁺) to cuprous ions (Cu¹⁺), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.     

Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins.

The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.     

Protein analysis of mammalian cells in monolayer culture using the bicinchoninic assay

We have applied the bicinchoninic acid (BCA) protein assay to rat brain primary astrocyte monolayer cultures growing in multiwell culture plates. The BCA method provides a more rapid and sensitive procedure with greater stability of color than is obtained using the Lowry method. Also, large numbers of samples can be read rapidly at the available wavelengths on an enzyme-linked immunosorbent assay microtiter plate reader. We found, however, artifactually high readings when using isotonic buffered sucrose to wash the cultures followed by sodium hydroxide to solubilize the cell protein. Such a procedure is commonly used for washing monolayer cell cultures in transport and binding studies. This effect was found to be due to hydrolysis of sucrose to the reducing sugar glucose. Use of Triton X-100 eliminated this problem, but this agent only solubilized about 80% of the protein that could be solubilized with sodium hydroxide. Furthermore, the high viscosity of Triton X-100 makes it more difficult to use. We found that washing the cells with isotonic mannitol solution followed by solubilization with sodium hydroxide gave reliable results. The sensitivity and speed of this method makes it suitable for multiple protein determinations in experiments using large numbers of cell culture samples.