Pandora box of BCA assay. Investigation of the accuracy and linearity of the microplate bicinchoninic protein assay: Analytical challenges and method modifications to minimize systematic errors

Bicinchoninic colorimetric assay is very widely used for total protein quantitative analysis. We report that bicinchoninic (BCA) total protein assay linearity range and the assay sensitivity are counterbalancing factors. BCA assay true linear range may be considerably narrower than the manufacturer’s advertised 20-2000 μg/ml and therefore the choice of the assay calibration range should not solely be dictated by the general recommendations of the user guide, but by the test specific needs and subsequent assay quality control. Expanding the BCA assay range up to 2000 μg/ml comes together with unavoidable heavy negative biases at low protein concentrations.
The negative bias at low protein concentration only exacerbates with longer incubation time and/or increased sample to working reagent ratio. To minimize the lack of accuracy at low protein concentration of a wide range BCA assay, we proposing an alternative approach: a two-step incubation and calibration. With a minimum of extra work, the two-step incubation/calibration approach is devoid of the standard BCA workflow disadvantages and biases.

Measuring the concentration of protein nanoparticles synthesized by desolvation method: comparison of Bradford assay, BCA assay, hydrolysis/UV spectroscopy and gravimetric analysis

The desolvation technique is one of the most popular methods for preparing protein nanoparticles for medicine, biotechnology, and food applications. We fabricated 11 batches of BSA nanoparticles and 2 batches of gelatin nanoparticles by desolvation method. BSA nanoparticles from 2 batches were cross-linked by heating at +70 °C for 2 h; other nanoparticles were stabilized by glutaraldehyde. We compared several analytical approaches to measuring their concentration: gravimetric analysis, bicinchoninic acid assay, Bradford assay, and alkaline hydrolysis combined with UV spectroscopy. We revealed that the cross-linking degree and method of cross-linking affect both Bradford and BCA assay.
Direct measurement of protein concentration in the suspension of purified nanoparticles by dye-binding assays can lead to significant (up to 50-60%) underestimation of nanoparticle concentration. Quantification of non-desolvated protein (indirect method) is affected by the presence of small nanoparticles in supernatants and can be inaccurate when the yield of desolvation is low. The reaction of cross-linker with protein changes UV absorbance of the latter. Therefore pure protein solution is an inappropriate calibrator when applying UV spectroscopy for the determination of nanoparticle concentration. Our recommendation is to determine the concentration of protein nanoparticles by at least two different methods, including gravimetric analysis.

Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods.

A rapid and highly sensitive bicinchoninic acid (BCA) reagent-based protein quantitation tool was developed using competitive resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) methods. A chelation reaction between BCA and Cu(+), which is reduced by protein in an alkaline environment, is exploited to create a BCA-Cu(+) complex that has strong RR and SERRS activities. Using these methods, protein concentrations in solutions can be quantitatively measured at concentrations as low as 50 μg mL(-1) and 10 pg mL(-1). There are many advantages of using RR and SERRS-based assays.
These assays exhibit a much wider linear concentration range and provide an additional one (RR method) to four (SERRS method) orders of magnitude increase in detection limits relative to UV-based methods. Protein-to-protein variation is determined using a reference to a standard curve at concentrations of BSA that exhibits excellent recoveries. These novel methods are extremely accurate in detecting total protein concentrations in solution. This improvement in protein detection sensitivity could yield advances in the biological sciences and medical diagnostic field and extend the applications of reagent-based protein assay techniques.

Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay.

  • Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease.
  • We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu(1+)-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction.
  • Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu(1+)-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration.

Protein rejecting properties of PEG-grafted nanoparticles: influence of PEG-chain length and surface density evaluated by two-dimensional electrophoresis and bicinchoninic acid (BCA)-proteinassay.

Poly (ethylene glycol) (PEG)-grafted nanoparticles have been described as potential intravenously injectable, long-circulating drug carriers. The in vivo behaviour of intravenous administered nanoparticles is decisively influenced by the interaction of the particles with the blood proteins. Two-dimensional electrophoresis (2-DE) was employed to study the protein rejecting properties of PEG-grafted polymer nanoparticles, possessing PEG-200 and PEG-400 chains, respectively.
The calculated PEG-chain distances varied between 0.39/0.31 nm (PEG-200) and 0.39/0.34 nm (PEG-400), therefore it was possible to study the influence of high chain densities attained by the use of short PEG chains on the protein adsorption. Apart from a stronger protein rejection of small-MW proteins achieved by PEG-chain distance diminution, the affinity of several proteins for the PEG-chains are shown and discussed. Beside the study of protein adsorption patterns, the total protein mass adsorbed to the particles, as well as the extent of protein desorption prior to 2-DE, was investigated using the bicinchoninic acid (BCA)-protein assay.

BCA Protein Assay Kit

20-abx293001 Abbexa
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BCA Protein Assay Kit

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BCA Protein Assay Kit

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BCA Protein Assay Kit

SK3021 Bio Basic 500Assays, 500preps 127.86 EUR

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BCA Protein Quantitation Kit

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BCA Protein Quantification Kit

E112-02 Vazyme 500 rxn 72.5 EUR

Protein Assay BCA Kit

06385-00 NACALAI TESQUE 1KIT 105 EUR

Mouse BLC + BCA 1 protein

30R-AB036 Fitzgerald 20 ug 312 EUR

BCA Protein Assay Kit II

K813-2500 Biovision each 210 EUR

BCA Protein Assay Kit II

K813-5000 Biovision each 307.2 EUR

Micro BCA Protein Assay Kit

18-444 Genesee Scientific 3,200 Micro-Assays/Unit 258.34 EUR

Micro BCA Protein Assay Kit

18-445 Genesee Scientific 3,200 Micro-Assays/Unit 265.8 EUR

Micro BCA Protein Assay Kit

AR1110 BosterBio 1kit 157.2 EUR

Micro BCA Protein Assay Kit

SK3061 Bio Basic 1250Assays, 1250preps 111.16 EUR

BCA Protein Assay Reagent A

20830016-1 Bio-WORLD 500 mL 161.88 EUR

Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE.

Measuring the protein content of a sample is a mandatory and frequently practiced procedure in the lab. Although the procedure is quite simple and convenient to perform with commercially available kits, incompatible reagents in the lysate can cause problems in the quality of measurement. Unfortunately these reagents are cornerstones of high efficiency lysing buffers, e.g. high amounts of urea or beta-mercaptoethanol. In this study we addressed the tolerance of the well-known BCA-assay (bicinchoninic acid) to various reagents in different concentrations, with special regard to a subsequent 2D-gelelectrophoresis.
As a result, the kit is incompatible with the recipes of regular 2D-buffers. Also, when mixing two different reagents interfering effects will occur in a non-predictable way. Therefore we established a new method to quantify protein content in lysates ready for 2D-gelelectrophoresis: by mixing an aliquot with SDS, an equilibration is performed to that the sample can be run on a regular 1D SDS PAGE. Image analysis following fluorescence staining (SYPRO Ruby) reveals the absolute protein content in comparison to a BSA dilution curve processed accordingly.

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