scholarly journals BCA Assay for protein quantification v1 (protocols.io.6n5hdg6)

protocols.io ◽  
2019 ◽  
Author(s):  
Karina Conkrite
Keyword(s):  
Protein Quantification ◽  
Bca Assay

scholarly journals A Ponceau S Staining-Based Dot Blot Assay for Rapid Protein Quantification of Biological Samples

Gels ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 43
Author(s):  
Dario Lucas Helbing ◽  
Leopold Böhm ◽  
Nova Oraha ◽  
Leonie Karoline Stabenow ◽  
Yan Cui
Keyword(s):  
Biological Samples ◽  
Protein Quantification ◽  
Dot Blot ◽  
Tissue Samples ◽  
Bicinchoninic Acid ◽  
Wide Range ◽  
Dot Blot Assay ◽  
Ponceau S ◽  
Commercial Kits ◽  
Bca Assay

Despite the availability of a wide range of commercial kits, protein quantification is often unreliable, especially for tissue-derived samples, leading to uneven loading in subsequent experiments. Here we show that the widely used Bicinchoninic Acid (BCA) assay tends to underestimate protein concentrations of tissue samples. We present a Ponceau S staining-based dot-blot assay as an alternative for protein quantification. This method is simple, rapid, more reliable than the BCA assay, compatible with biological samples lysed in RIPA or 2x SDS gel-loading buffer, and also inexpensive.

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

2020 ◽  
Vol 608 ◽  
pp. 113904 ◽  
Author(s):  
Javiera Cortés-Ríos ◽  
Ana María Zárate ◽  
Juan David Figueroa ◽  
Joaquín Medina ◽  
Eduardo Fuentes-Lemus ◽  
...  
Keyword(s):  
Protein Quantification ◽  
Bicinchoninic Acid ◽  
Bca Assay

Advantages of the KDS/BCA Assay over the Bradford Assay for Protein Quantification in White Wine and Grape Juice

2014 ◽  
Vol 66 (2) ◽  
pp. 227-233 ◽  
Author(s):  
Diana Gazzola ◽  
Simone Vincenzi ◽  
Gabriella Pasini ◽  
Giovanna Lomolino ◽  
Andrea Curioni
Keyword(s):  
Grape Juice ◽  
White Wine ◽  
Protein Quantification ◽  
Bradford Assay ◽  
Bca Assay

scholarly journals A Ponceau Staining based Dot-Blot Assay for reliable and cost-effective Protein Quantification

2019 ◽  
Author(s):  
Dario-Lucas Helbing ◽  
Leopold Böhm ◽  
Yan Cui ◽  
Leonie Karoline Stabenow ◽  
Helen Morrison
Keyword(s):  
Protein Extraction ◽  
Cost Effective ◽  
Protein Quantification ◽  
Dot Blot ◽  
Effective Manner ◽  
Dot Blot Assay ◽  
Ponceau S ◽  
Reliable Quantification ◽  
Complete Protein ◽  
Bca Assay

AbstractReliable quantification of protein extracts from tissues can be a challenge e.g. due to interference of the high fat content in tissues of the nervous system. Further problems like under- or overerstimation of protein concentrations in protein quantification kits like the bicinchoninic acid (BCA) assay can occur. In addition, common lysis buffers such as RIPA buffer are known to be unable to solubilize a large amount of proteins (~10-30%) leading to unsatisfactory and unreliable experimental results with techniques such as immunoblotting. In this work, we have developed a Ponceau S staining based protein quantification assay. This assay is compatible with tissues or cells directly lysed in 2x SDS gel loading buffer, containing bromophenolblue, leading to more complete protein extraction. Protein concentrations of several samples can be determined in a fast and cost-effective manner and subsequent experiments (e.g. Western blot) can be performed without loss of proteins. The presented protein quantification method is highly reliable, fast and economical. Using this method, it is possible to save between 2300 to 3200€ per 1000 lysates as compared to the costs of a commercial BCA kit.

scholarly journals Comparative Analysis of Protein Quantification Methods for the Rapid Determination of Protein Loading in Liposomal Formulations

Pharmaceutics ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 39 ◽  
Author(s):  
Maryam Hussain ◽  
Neil Forbes ◽  
Yvonne Perrie
Keyword(s):  
Small Molecules ◽  
High Performance ◽  
Rapid Determination ◽  
Correlation Coefficients ◽  
Cationic Liposome ◽  
Protein Quantification ◽  
Protein Loading ◽  
Rp Hplc ◽  
Evaporative Light Scattering ◽  
Bca Assay

Advances in manufacturing processes provide the ability for the high throughput production of liposomes containing a range of moieties, from small molecules to large biologicals (including proteins and nucleic acids for prophylactic and therapeutic applications). Whilst rapid quantification methods for small molecules are generally well established, the ability to rapidly quantify liposomal entrapment of proteins is limited. Indeed, most standard protein quantification techniques (including the BCA assay and Reverse phase-high performance liquid chromatography (RP-HPLC)) measure protein encapsulation indirectly, by measuring the amount of non-incorporated drug, and subtracting from the initial amount of protein added. However, this can give inaccurate and misrepresentative results. To address this, we have developed a range of methods to directly quantify protein entrapment within liposomes. The encapsulation efficiency within neutral, anionic and cationic liposome formulations was determined by three techniques; BCA assay, RP-HPLC and HPLC coupled to an evaporative light scattering detector, (HPLC-ELSD). All three methods are reliable for the quantification of protein, with linear responses and correlation coefficients of 0.99, and LOQ for all three methods being less than 10 µg/mL. Here within, we provide three methods for the rapid and robust quantification of protein loading within liposomal (and other bilayer) vesicle systems.

scholarly journals Towards an SI-Traceable Reference Measurement System for Seven Serum Apolipoproteins Using Bottom-Up Quantitative Proteomics: Conceptual Approach Enabled by Cross-Disciplinary/Cross-Sector Collaboration

2020 ◽  
Author(s):  
Christa M Cobbaert ◽  
Harald Althaus ◽  
Ilijana Begcevic Brkovic ◽  
Uta Ceglarek ◽  
Stefan Coassin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cardiovascular Disease ◽  
Measurement System ◽  
Protein Quantification ◽  
Test Results ◽  
Conceptual Approach ◽  
Reference Measurement ◽  
Lipid Panel ◽  
Mass Spectrometry Technology ◽  
Reference Measurement System

Abstract Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

scholarly journals Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gianluca Trinco ◽  
Valentina Arkhipova ◽  
Alisa A. Garaeva ◽  
Cedric A. J. Hutter ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Kinetic Mechanism ◽  
Protein Quantification ◽  
Detergent Solution ◽  
Sodium Ions ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Uptake Rates ◽  
Bona Fide ◽  
Inside Out

AbstractIt is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

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