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.

Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times

Amongst the available methodologies for protein determination, the bicinchoninic acid (BCA) assay highlights for its simplicity, sensitivity, repeatability and reproducibility. Nevertheless, in spite that the general principle behind this methodology is known, there are still unanswered questions regarding the chemistry behind the assay and the experimental conditions commonly employed. The present work explored the kinetics, and the analytical response of the assay to free amino acids, peptides (containing tryptophan and tyrosine), and proteins. Results revealed kinetic profiles characterized by the absence of plateaus, with behaviors depending on the type of the sample.
The latter, along with contribution to the BCA index elicited by oxidation products generated at the side chain of tryptophan and tyrosine, as well as pre-oxidized β-casein, evidenced the presence of complex reaction mechanisms. In spite of such complexity, our results showed that the BCA index is not modulated by the incubation time. This applies for responses producing absorbance intensities (at 562 nm) higher than 0.1. Therefore, we propose that the assay can be applied at shorter incubation times (15 min) than those indicated in manufactures specifications, and usually used by researches and industry (30 min at 37ºC).

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.

Pre-diluted BSA (bovine serum albumin) standards (1 set) for BCA Optima Protein Assay kit

BCA-BSA-1 1 pk 196.8 EUR

Pre-diluted BSA (bovine serum albumin) standards (750 ug/ml) for BCA Optima Protein Assay kit

BCA-BSA-750 1 ml 270 EUR

Pre-diluted BSA (bovine serum albumin) standards (250, 125, 62.5, 31.25, and 0.0 ug/ml) for BCA Optima Protein Assay kit

BCA-BSA-2 1 pk 196.8 EUR

BCA

HY-15908 500mg 129.6 EUR

Pre-diluted BGG (bovine gamma globulin) standards (1 set: 2000, 1000, 500, 125, 62.5, 31.25, and 0 ng/ml BGG) for BCA Optima Protein Assay kit

BCA-BGG-1 1 pk 196.8 EUR

BCA Protein Quantification Kit

E112-01 250 rxn 158.4 EUR

BCA Protein Quantification Kit

E112-02 500 rxn 182.4 EUR

BCA Protein Quantitation Kit

55R-1544 1000 assays 457.2 EUR

BCA Protein Assay Kit

20-abx090640
  • 266.40 EUR
  • 322.80 EUR
  • 200 tests
  • 500 tests

BCA Protein Assay Kit

abx098126-100ml 100 ml 303.6 EUR

BCA Protein Assay Kit

20-abx293001
  • 284.40 EUR
  • 811.20 EUR
  • 435.60 EUR
  • 100 tests
  • 2500 tests
  • 500 tests

BCA Protein Assay Kit

AR0146 1kit 121.2 EUR

BCA Protein Assay Kit

AR0146-1L 1kit 547.2 EUR

BCA Protein Assay Kit

AR0146-500 1kit 339.6 EUR

BCA Protein Quantitation Kit

K2185-1000 1000 assays 385.2 EUR

BCA Protein Assay Kit

SK3021 500Assays, 500preps 127.86 EUR

BCA Protein Quantitation Kit

K812-1000 each 352.8 EUR

BCA Reagent, 16ML

C144-16ML 16ML 195.6 EUR

BCA Enhancer, 320UL

C145-320UL 320UL 195.6 EUR

BCA disodium salt

2810-100G each 1038 EUR

BCA disodium salt

2810-1G each 144 EUR

BCA disodium salt

2810-500G each 3126 EUR

BCA disodium salt

2810-5G each 261.6 EUR

Mouse BLC + BCA 1 protein

30R-AB036 20 ug 327.6 EUR

BCA Protein Assay Kit (Enhanced)

20-abx090642
  • 266.40 EUR
  • 340.80 EUR
  • 200 tests
  • 500 tests

BCA-1 / BLC (CXCL13) Protein

20-abx260661
  • 4101.60 EUR
  • 393.60 EUR
  • 276.00 EUR
  • 1 mg
  • 20 ug
  • 5 ug

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.

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