Quantitation of submicrogram quantities of protein by an improved protein-dye binding assay

doi:10.1016/0005-2795(78)90398-7

Biochimica et Biophysica Acta (BBA) - Protein Structure

Volume 533, Issue 2, 26 April 1978, Pages 525-529

https://doi.org/10.1016/0005-2795(78)90398-7 Get rights and content

Abstract

A quantitative assay method for protein is described which is based on the color change occurring in Coomassie Brilliant Blue G-250 when it binds to protein. This modification of two similar procedures further increases the sensitivity, simplicity, and stability of the protein-dye binding assay over those of other commonly used assays for protein.

References (9)

M.M. Bradford
Anal. Biochem.
(1976)
J.J. Sedmak et al.
Anal. Biochem.
(1977)
A.H. Reisner et al.
Anal. Biochem.
(1975)
O.H. Lowry et al.
J. Biol. Chem.
(1951)

There are more references available in the full text version of this article.

Cited by (409)

Salivary GFAP as a potential biomarker for diagnosis of mild cognitive impairment and Alzheimer's disease and its correlation with neuroinflammation and apoptosis
2021, Journal of Neuroimmunology
Citation Excerpt :
After thawing, samples are centrifuged to remove any formed aggregates, and analyses are conducted at the soluble part of the samples. The protein content of the samples was analyzed with the Bradford-Bearden assay, as previously described (Bearden, 1978). Due to variations in the protein content of saliva samples (Table S1), all results were finally normalized versus mg of total protein of each saliva sample, to avoid variability in results due to this factor.
Glial fibrillary acidic protein (GFAP) is the main constituent of the astrocytic cytoskeleton, overexpressed during reactive astrogliosis–a hallmark of Alzheimer's Disease (AD). GFAP and established biomarkers of neurodegeneration, inflammation, and apoptosis have been determined in the saliva of amnestic-single-domain Mild Cognitive Impairment (MCI) (Ν = 20), AD (Ν = 20) patients, and cognitively healthy Controls (Ν = 20). Salivary GFAP levels were found significantly decreased in MCI and AD patients and were proven an excellent biomarker for discriminating Controls from MCI or AD patients. GFAP levels correlate with studied biomarkers and Aβ₄₂, IL-1β, and caspase-8 are its main predictors.
Lipase-active heterogeneous biocatalysts for enzymatic synthesis of short-chain aroma esters
2021, Biocatalysis and Agricultural Biotechnology
Citation Excerpt :
For genetic engineering manipulations the following equipment were used: Gene Pulser Xcell Total System Electroporator (BioRad, USA), Unimax 2010 orbital shaker (Heidolph, Gemany), DNA Thermal Cycler BIS (RF), Gel-Pro analyzer (RF), J2-21 centrifuge (Beckman, USA), etc. The concentrations of rPichia/lip were measured spectrophotometrically by following methods: 1) UV absorption at a wavelength of 280 nm, taking into account its molar extinction coefficient (36280 L mol−1·cm−1), 2) VIS Bearden’ method using Coomassie G-250 dye (Bearden, 1978), 3) densitometry of electropherogram band stained with Coomassie brilliant blue in a gel after SDS-PAAG versus standard human serum albumin titration bands using Gel-Pro analyzer. Texture parameters of the adsorbents used for the lipase immobilization were studied by N2-adsorption and Hg-intrusion porosimetry using AutoPore 9200 and ASAP 2400 V3.07 setups (Micromeritics Instrument Corporation, USA).
The lipase-active heterogeneous biocatalysts were prepared by adsorptive immobilization of Thermomyces lanuginosus lipase (designated as rPichia/lip) produced by authoring recombinant Pichia pastoris strain. Biocatalysts of LipoCarb type were prepared by spontaneous adsorption of rPichia/lip on the authoring macroporous carbon aerogel. Biocatalysts of LipoSil type were prepared by forced adsorption of rPichia/lip on mesoporous silica. The prepared biocatalysts were studied carefully in the esterification of short-chain fatty acids (butyric C4, C5–C7) and aliphatic alcohols (ethanol C2, C3–C5, C8) in order to produce valuable fruity-smelling esters. Biocatalytic properties, such as enzymatic activity, substrates specificity, and operational stability, were found to depend on chemical nature of both the adsorbents and organic solvents, primarily on their polarity. When comparing the activity in the synthesis of n-butyl heptanoate, it was found that the LipoCarb biocatalysts demonstrated activity an order of magnitude higher than LipoSil type. The Michaelis-Menten kinetic constants differed by a factor of 30, e.g. 1.1·10² s⁻¹ and 3.7 s⁻¹, respectively. The operational stability of the lipase-active biocatalysts was sufficiently high; rPichia/lip adsorbed on carbon aerogel retained till 96% of the initial activity after 29 reaction cycles, whereas rPichia/lip adsorbed on silica completely retained the activity after 38 reaction cycles of esterification under optimal reaction conditions selected. The prepared biocatalysts of LipoCarb and LipoSil types were promising for the Green enzymatic synthesis of fragrance and aroma esters under very mild ambient conditions (20±2 °C, 1 bar).
Respiratory burst oxidases and apoplastic peroxidases facilitate ammonium syndrome development in Arabidopsis
2021, Environmental and Experimental Botany
Ammonium-nitrogen (NH₄⁺) nutrition is linked to metabolic over-reduction for plants. The characteristic symptom of sole NH₄⁺ nutrition is growth suppression, signifying this condition as the ammonium syndrome. In the present study, we investigated the mechanism of perception of high NH₄⁺ conditions in Arabidopsis thaliana plants by examining apoplastic reactive oxygen species (ROS) metabolism. Major enzyme activity and a special pattern of expression of NADPH-dependent respiratory burst oxidases (RBOH) was found in Arabidopsis individuals cultured under NH₄⁺ as the sole nitrogen source. This oxidative burst is independent of RBOHD/F expression and does not activate typical intracellular signalling pathways. In addition, elevated superoxide dismutase and apoplastic secretory peroxidase activities contributed to hydrogen peroxide (H₂O₂) accumulation in plants exposed to NH₄⁺ nutrition. Consequently, higher H₂O₂ contents were determined in the extracellular space and were localised cytochemically. H₂O₂ is a substrate for cell wall cross-linking peroxidases, which showed enhanced activity in the presence of NH₄⁺. Increase of cell wall polymerisation, could in turn inhibit cell elongation and slow down growth, as observed under NH₄⁺ toxicity.
Studying RNA-protein interactions of pre-mRNA complexes by mass spectrometry
2015, Methods in Enzymology
RNA–protein interactions play a crucial role in gene expression. These interactions take place in so-called ribonucleoprotein (RNP) complexes. To investigate which proteins interact with RNA in these complexes, and how they do so, UV-light-induced cross-linking has proven to be a valuable, yet straightforward technique. UV irradiation induces a covalent bond between the RNA and the proteins, whereafter cross-linked proteins can be identified by mass spectrometric (MS) approaches. Moreover, the cross-linked region of the protein, and often the actual cross-linked amino acid, can be identified by state-of-the-art MS, as can the cross-linked RNA moiety. This protocol describes in detail how to isolate peptide–RNA oligonucleotide cross-links from UV-irradiated human pre-mRNA RNPs and to perform the subsequent MS investigation of these peptide–RNA conjugates in combination with a dedicated computational analysis, in order to obtain sequence information about the cross-linked peptide and oligoribonucleotide. The described workflow can be applied to any RNP, irrespective of its origin, e.g., RNPs assembled in vitro (as described here) or RNPs isolated from UV-irradiated cells, either ex vivo or in vivo.
Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation
2024, International Journal of Molecular Sciences
In vitro and in silico evaluation of the serrapeptase effect on biofilm and amyloids of Pseudomonas aeruginosa
2023, Applied Microbiology and Biotechnology

View all citing articles on Scopus

View full text

BBA reportQuantitation of submicrogram quantities of protein by an improved protein-dye binding assay

Abstract

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

BBA report
Quantitation of submicrogram quantities of protein by an improved protein-dye binding assay