Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Brady, Pamlea N.; Macnaughtan, Megan A.

doi:10.1016/j.ab.2015.08.027

Cited by 42 publications

(33 citation statements)

References 63 publications

(56 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cu 1+ forms a complex with BCA, resulting a colored water-soluble chelate [2]. The intensity of the color change by these assays is measured by absorbance photometry at 595 nm and 562 nm for the Bradford and BCA assays respectively [4]. Both methods allow the detection of proteins in μg/mL range.…”

Section: Introductionmentioning

confidence: 99%

Application of BisANS fluorescent dye for developing a novel protein assay

et al. 2019

View full text Add to dashboard Cite

In many biology- and chemistry-related research fields and experiments the quantification of the peptide and/or protein concentration in samples are essential. Every research environment has unique requirements, e.g. metal ions, incubation times, photostability, pH, protease inhibitors, chelators, detergents, etc. A new protein assay may be adequate in different experiments beyond or instead of the well-known standard protocols (e.g. Qubit, Bradford or bicinchoninic acid) in related conceptions. Based on our previous studies, we developed a novel protein assay applying the 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (BisANS) fluorescent dye. This molecule has several advantageous properties related to protein detection: good solubility in water, high photostability at adequate pH, quick interaction kinetics (within seconds) with proteins and no exclusionary sensitivity to the chelator, detergent and inhibitor ingredients. The protocol described in this work is highly sensitive in a large spectrum to detect protein (100-fold diluted samples) concentrations (from 0.28 up to more than 100 μg/mL). The BisANS protein assay is valid and applicable for quantification of the amount of protein in different biological and/or chemical samples.

show abstract

Section: Introductionmentioning

confidence: 99%

Application of BisANS fluorescent dye for developing a novel protein assay

et al. 2019

View full text Add to dashboard Cite

show abstract

“…[1][2][3][4] This method was proposed by Marion M. Bradford in 1976 5 and relies on the interaction of Comassie Brilliant Blue G-250 dye with the protein. After a short-time incubation, the anionic product of such reaction is optically monitored at 595 nm.…”

Section: Introductionmentioning

confidence: 99%

Smartphone for Point-of-Care Quantification of Protein by Bradford Assay

Camargo¹,

Vicentini²,

Gobbi³

et al. 2016

J. Braz. Chem. Soc.

View full text Add to dashboard Cite

We address in this paper a powerful point-of-care platform to conduct the Bradford assay. Our method was based on smartphone for colorimetric quantification of total protein in human blood plasma, presenting low cost, simplicity, portability, autonomy and ability for remote transmission of the data. Other advantage concerns the high number of smartphone's users worldwide. This feature contributes for the application of the method by non-specialist people. The interferences from external light were successfully solved by illuminating the samples with a straightforward negatoscope. Our method generated a satisfactory exactness, with accuracy percentages ranging from 87.2 to 99.1%. Keywords: rapid test, albumin, human plasma, colorimetry, negatoscope IntroductionBradford assay is one of the most usual methods applied for determining protein in solution. [1][2][3][4] This method was proposed by Marion M. Bradford in 1976 5 and relies on the interaction of Comassie Brilliant Blue G-250 dye with the protein. After a short-time incubation, the anionic product of such reaction is optically monitored at 595 nm. 6 The advantages of the Bradford assay are that its routine is user-friendly, fast, reproducible, sensitive and selective to the target molecule. 7,8 Conversely, some pharmaceutical excipients interfere on the response of this approach even at low contents. In this case, the precipitation of the protein with sucrose showed to be an efficient alternative.9 Other bottleneck concerns the poor portability and relatively high cost (ca. US$ 10,000.00) of the spectrophotometer applied in the Bradford assays. These features undermine the compatibility of the method toward point-of-care (POC) analyses. 10POC platforms present low cost and high portability, robustness and simplicity. Accordingly, such technologies bypass the necessity for specialized personal and allow in situ measurements even at resource-limited environments, presenting key social and economic implications. Such methods have been recently used at diverse applications. [11][12][13][14][15][16] The POC technology is the major segment of the global in vitro diagnostics market. 11Nonetheless, despite the advantages of these methods, only a few commercial executions had been observed because of the production cost of such analytical platforms. Such drawback inhibits the creation of profitable businesses. 17We address herein a POC platform for performing the Bradford assay using smartphone-based optical detection. The smartphones have been widely used to the development of POC analytical tools for different colorimetric quantitative analyses, including immunoassays, biochemical and healthcare experiments. [18][19][20][21][22] These applications are possible because the smartphones present the two functional components necessary for accomplishing optical detection: source (light-emitting diode, LED, white light from flash) and radiation detector (digital camera). 23 This camera exhibits resolution and sensitivity greater than those of conventional phone camera...

show abstract

“…The Folin–phenol reagent consists of phosphomolybdic–phosphotungstic acid, which is reduced to a blue-colored solution by protein in an alkaline Cu 2+ tartrate solution [ 105 , 107 ], whereas the color in the Bradford assay is formed due to complex formation between the protein and the Coomassie blue G-250 dye. Under acidic conditions, the protonated red dye is transformed to an anionic blue form through a dye–protein electrostatic and hydrophobic interaction [ 108 , 109 ].…”

Section: Characterization Of Fucoidan Qualitymentioning

confidence: 99%

Fucoidan Characterization: Determination of Purity and Physicochemical and Chemical Properties

et al. 2020

View full text Add to dashboard Cite

Fucoidans are marine sulfated biopolysaccharides that have heterogenous and complicated chemical structures. Various sugar monomers, glycosidic linkages, molecular masses, branching sites, and sulfate ester pattern and content are involved within their backbones. Additionally, sources, downstream processes, and geographical and seasonal factors show potential effects on fucoidan structural characteristics. These characteristics are documented to be highly related to fucoidan potential activities. Therefore, numerous chemical qualitative and quantitative determinations and structural elucidation methods are conducted to characterize fucoidans regarding their physicochemical and chemical features. Characterization of fucoidan polymers is considered a bottleneck for further biological and industrial applications. Consequently, the obtained results may be related to different activities, which could be improved afterward by further functional modifications. The current article highlights the different spectrometric and nonspectrometric methods applied for the characterization of native fucoidans, including degree of purity, sugar monomeric composition, sulfation pattern and content, molecular mass, and glycosidic linkages.

show abstract

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Cited by 42 publications

References 63 publications

Application of BisANS fluorescent dye for developing a novel protein assay

Application of BisANS fluorescent dye for developing a novel protein assay

Smartphone for Point-of-Care Quantification of Protein by Bradford Assay

Fucoidan Characterization: Determination of Purity and Physicochemical and Chemical Properties

Contact Info

Product

Resources

About