Correction in Bicinchoninic Acid (BCA) Absorbance Assay to Analyze Protein Concentration

Nano LIFE ◽  
2018 ◽  
Vol 08 (03) ◽  
pp. 1850005 ◽  
Author(s):  
Daniel L. Smith ◽  
Elizabeth N. Lemieux ◽  
Sutapa Barua
Keyword(s):  
Incubation Time ◽  
Protein Concentration ◽  
Coupling Reaction ◽  
Careful Analysis ◽  
Accurate Analysis ◽  
Formation Reaction ◽  
Bicinchoninic Acid ◽  
Microparticle Formation ◽  
Polymer Microparticles ◽  
Bca Assay

Conducting the bicinchoninic acid (BCA) assay directly after a coupling reaction using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and [Formula: see text]-hydroxysuccinimide (NHS) chemistry produces significant errors. Here we present a correction for the quantification of gelatin in the supernatant (SN) following gelatin conjugation to polymer microparticles using EDC and NHS chemistry. Following the conjugation reaction, SNs from the gelatin-microparticle formation reaction are treated with BCA assay reagents and quantified for the percentage of unbound gelatin in the solution. NHS was found to interfere with the BCA assay reagents and is dependent on incubation time. It is found that the large concentration (500[Formula: see text][Formula: see text]g/mL) of NHS in the conjugation reaction interferes with the sensitivity of gelatin present in SNs. The interference from NHS requires a careful analysis to distinguish the BCA background absorbance from the sample absorbance. Using an NHS control solution can correct NHS interference and thus decrease the expensive iterations in gelatin quantification and enable accurate analysis of gelatin content. The accuracy of gelatin quantification is further improved by reducing the BCA assay incubation time to approximately 20[Formula: see text]min, compared with the recommended 30[Formula: see text]min. This re-assessment of BCA assay is important to avoid misestimating biases in bioconjugation processes.

Left Ventricular Hypertrophy

2014 ◽  
Vol 19 (2) ◽  
pp. 11-15
Author(s):  
Steven L. Demeter
Keyword(s):  
Risk Factors ◽  
Left Ventricular Hypertrophy ◽  
Ventricular Hypertrophy ◽  
Left Ventricular Mass Index ◽  
Medical Science ◽  
Careful Analysis ◽  
Left Ventricular ◽  
Hypertensive Disease ◽  
Accurate Analysis ◽  
Clear Explanation

Abstract The fourth, fifth, and sixth editions of the AMA Guides to the Evaluation of Permanent Impairment (AMA Guides) use left ventricular hypertrophy (LVH) as a variable to determine impairment caused by hypertensive disease. The issue of LVH, as assessed echocardiographically, is a prime example of medical science being at odds with legal jurisprudence. Some legislatures have allowed any cause of LVH in a hypertensive individual to be an allowed manifestation of hypertensive changes. This situation has arisen because a physician can never say that no component of LVH was not caused by the hypertension, even in an individual with a cardiomyopathy or valvular disorder. This article recommends that evaluators consider three points: if the cause of the LVH is hypertension, is the examinee at maximum medical improvement; is the LVH caused by hypertension or another factor; and, if apportionment is allowed, then a careful analysis of the risk factors for other disorders associated with LVH is necessary. The left ventricular mass index should be present in the echocardiogram report and can guide the interpretation of the alleged LVH; if not present, it should be requested because it facilitates a more accurate analysis. Further, if the cause of the LVH is more likely independent of the hypertension, then careful reasoning and an explanation should be included in the impairment report. If hypertension is only a partial cause, a reasoned analysis and clear explanation of the apportionment are required.

scholarly journals Oat Protein Concentrates with Improved Solubility Produced by an Enzyme-Aided Ultrafiltration Extraction Method

Foods ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3050
Author(s):  
Mika Immonen ◽  
Julia Myllyviita ◽  
Tuula Sontag-Strohm ◽  
Päivi Myllärinen
Keyword(s):  
Response Surface Methodology ◽  
G Protein ◽  
Incubation Time ◽  
Extraction Method ◽  
Protein Concentration ◽  
Treatment Process ◽  
Alkaline Ph ◽  
Protein Concentrates ◽  
Pre Treatment ◽  
Enzyme Dosage

The aim of this study was to develop an extraction method to produce highly functional oat protein concentrates. We investigated the possibility of combining enzyme-aided slightly alkaline (pH 8.0) extraction with ultrafiltration and subsequent diafiltration for concentration of the extracted oat proteins. A further aim was to study how the deamidation of oat proteins with protein-glutaminase (PG) improves the solubility of proteins as a function of the following parameters: pH (6.0–9.0), enzyme dosage (4–20 U/g protein), and incubation time (1–4 h) with response surface methodology (RSM). Furthermore, we investigated selected functional properties, such as heat-induced gelation and solubility, of the oat protein concentrates. The chosen parameters for the enzymatic deamidation pre-treatment process by PG were as follows: pH 8.0, dosage 11.0 U/g protein, and an incubation time of 4 h (1 h at native pH and 3 h at pH 8.0). Two oat protein concentrates were produced, non-deamidated and ultrafiltered, and deamidated and ultrafiltered, with protein concentrations of 45.0 and 52.4%, respectively. The solubility of both oat protein concentrates was significantly improved at neutral and slightly alkaline pH compared to the solubility of proteins extracted from the starting material. Additionally, both oat protein concentrates produced equally strong heat-induced gel-like structures at a protein concentration of 10%.

Oxidative Intramolecular C-C Bond Formation Reactions of 1,2-Diarylbenzenes: Syntheses of Highly Conjugated Double-Bridged Polycyclic Aromatic Hydrocarbons

Synthesis ◽  
2021 ◽  
Author(s):  
Md. Rafikul Islam ◽  
Tohru Nishinaga ◽  
Kazunori Hirabayashi ◽  
Toshio Shimizu ◽  
Ken-ichi Sugiura
Keyword(s):  
Theoretical Calculations ◽  
Bond Formation ◽  
Cation Radical ◽  
Coupling Reaction ◽  
Oxidation Reactions ◽  
Marked Contrast ◽  
Radical Species ◽  
Formation Reaction ◽  
Polycyclic Aromatic ◽  
Spin Densities

Oxidation reactions of 1,2-diarylbenzenes induce intramolecular C-C bond formation. The substrates studied here were prepared by the stepwise Suzuki-Miyaura coupling reaction that introduced 2-naphthyl, 2-anthranyl, and 2-pyrenyl groups on the ortho positions of benzene. The subsequent oxidation reaction with FeCl3 induced an oxidative C-C bond formation reaction in the interior regions of the molecules. In marked contrast to our previous observations, two C-C bonds were formed. Theoretical calculations indicated that large spin densities at the reaction positions of the bis(cation radical) and/or cation radical species are needed for the C-C bond formation. The π-expanded molecules obtained here showed bathochromic shifts in the absorption spectra and amphoteric multi-redox behavior in electrochemistry.

scholarly journals Silver-Catalyzed Decarboxylative Cross-coupling Radical Reaction: Direct Synthesis of Coenzyme Q Compounds

2019 ◽  
Author(s):  
Wan-Yue Luo ◽  
Yan Zhao ◽  
Tian-Li Zhang ◽  
Jin Wang ◽  
Xiao Hu
Keyword(s):  
Coenzyme Q ◽  
Direct Synthesis ◽  
Radical Reaction ◽  
Coupling Reaction ◽  
Cross Coupling ◽  
Formation Reaction ◽  
Cross Coupling Reaction ◽  
Direct Cross ◽  
Aliphatic Carboxylic Acids ◽  
General Method

<p>An efficient and general method for the synthesis of Coenzyme Q compounds through the activation of 1,4-benzoquinone C<i><sub>sp2</sub></i>-H bond has been developed. This C-C bond formation reaction proceeds readily in an open flask by direct cross-coupling reaction of Coenzyme Q<sub>0</sub> with commercially available aliphatic carboxylic acids utilizing AgNO<sub>3</sub> as catalyst and K<sub>2</sub>S<sub>2</sub>O<sub>8</sub> as oxidant in aqueous solution. This radical reaction is operationally simple and amenable to gram-scale synthesis. <sub></sub></p>

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.

scholarly journals BIOCHEMICAL ANALYSES OF SEED EXUDATES DURING EARLY IMBIBITION

HortScience ◽  
1992 ◽  
Vol 27 (6) ◽  
pp. 684g-685
Author(s):  
Eric E. Roos ◽  
Sharon Sowa ◽  
Kristin Pedas
Keyword(s):  
Oryza Sativa ◽  
Protein Concentration ◽  
Room Temperature ◽  
Seed Viability ◽  
Distilled Water ◽  
Short Term ◽  
Time Period ◽  
High Viability ◽  
Biochemical Analyses ◽  
Bca Assay

Short term soaking of seeds does not appear to be detrimental to seed viability and may provide a means of testing seed viability non-destructively. Seeds of corn (Zea mays L.) and rice (Oryza sativa L.), differing in viability, were soaked for 0.5, 1, 2 and 4 hr in distilled water at room temperature. Analyses of pH, protein/polypeptides (BCA assay and absorbance at 280 nm), and potassium (and other metals), were done on individual seed leachates. After each time period seeds were germinated for 7 d to determine viability. For both corn and rice, pH remained constant between 0.5 and 4 hr of soaking. Protein concentration gradually increased during the 4 hr soak in both corn and rice, but varied with seed lot. Potassium was the most common metal excreted and increased 3 to 4 fold between 0.5 and 4 hr of soaking. Although seed to seed variability in any given lot was high, in general, low viability seeds lost more cellular constituents than high viability seeds.

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