Measuring Protein Concentration with Absorbance, Lowry, Bradford Coomassie Blue, or the Smith Bicinchoninic Acid Assay Before Electrophoresis

2018 ◽  
pp. 31-39 ◽  
Author(s):  
J. P. Dean Goldring
Keyword(s):  
Protein Concentration ◽  
Bicinchoninic Acid ◽  
Coomassie Blue

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.

Albumin-Induced Endocytic Vacuoles in Tetrahymena Pyrijormis

1975 ◽  
Vol 33 ◽  
pp. 694-695
Author(s):  
L. J. Brenner ◽  
D. G. Osborne ◽  
B. L. Schumaker
Keyword(s):  
Electron Microscopy ◽  
Bovine Serum Albumin ◽  
Bovine Serum ◽  
Protein Concentration ◽  
Tetrahymena Pyriformis ◽  
Sequence Of Events ◽  
Cytoplasmic Bodies ◽  
Food Vacuoles ◽  
Mouse Ascites ◽  
Normal Human

Exposure of the ciliate, Tetrahymena pyriformis, strain WH6, to normal human or rabbit sera or mouse ascites fluids induces the formation of large cytoplasmic bodies. By electron microscopy these (LB) are observed to be membrane-bounded structures, generally spherical and varying in size (Fig. 1), which do not resemble the food vacuoles of cells grown in proteinaceous broth. The possibility exists that the large bodies represent endocytic vacuoles containing material concentrated from the highly nutritive proteins and lipoproteins of the sera or ascites fluids. Tetrahymena mixed with bovine serum albumin or ovalbumin solutions having about the same protein concentration (7g/100 ml) as serum form endocytic vacuoles which bear little resemblance to the serum-induced LB. The albumin-induced structures (Fig. 2) are irregular in shape, rarely spherical, and have contents which vary in density and consistency. In this paper an attempt is made to formulate the sequence of events which might occur in the formation of the albumin-induced vacuoles.

Monoamine Oxidase and Other Mitochondrial Enzymes in Density Subpopulations of Human Platelets

1988 ◽  
Vol 59 (01) ◽  
pp. 029-033 ◽  
Author(s):  
K G Chamberlain ◽  
D G Penington
Keyword(s):  
Monoamine Oxidase ◽  
Protein Concentration ◽  
Close Correlation ◽  
Human Platelets ◽  
Mitochondrial Enzymes ◽  
Density Fractions ◽  
Percoll Gradients ◽  
Normal Human ◽  
The Mean ◽  
Very High

SummaryNormal human platelets have been separated according to density on continuous Percoll gradients and the platelet distribution divided into five fractions containing approximately equal numbers of platelets. The mean volumes and protein contents of the platelets in each fraction were found to correlate positively with density while the protein concentration did not differ significantly between the fractions. Four mitochondrial enzymes (monoamine oxidase, glutamate dehydrogenase, cytochrome oxidase and NADP-dependent isocitrate dehydrogenase) were assayed and their activities per unit volume were found to increase in a very similar monotonie fashion with platelet density. When MAO and GDH were assayed on the same set of density fractions the correlation between the two activities was very high (r = 0.94–1.00, p <0.001) and a similar close correlation was found between MAO and ICDH. The results support the hypothesis that high density platelets either have a higher concentration of mitochondria or have larger mitochondria than low density platelets.

Does Low Protein Concentration of Tissue-type Plasminogen Activator Predict a Low Risk of Spontaneous Deep Vein Thrombosis?

1995 ◽  
Vol 74 (02) ◽  
pp. 718-721 ◽  
Author(s):  
Jørgen Gram ◽  
Johannes Sidelmann ◽  
Jørgen Jespersen
Keyword(s):  
Deep Vein Thrombosis ◽  
Confidence Interval ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Protein Concentration ◽  
Tissue Type ◽  
Vein Thrombosis ◽  
Low Protein ◽  
Deep Vein ◽  
Pai 1

SummaryMany reports have demonstrated an abnormal fibrinolysis in a subset of patients with deep vein thrombosis. We have studied systemic global fibrinolytic activity and protein concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma of 25 young patients with a previous instance of spontaneous deep vein thrombosis documented by phlebography and in 50 healthy controls. The two populations were comparable with respect to a number of base-line variables (age, height, weight, etc.), while the patients had significantly lower fibrinolytic activity (p <0.02), and significantly higher protein concentrations of t-PA (p <0.0001) and PAI-1 (p <0.0006).We used probit scale plots to identify the consequence of different cut-off points to separate patients from controls. Reasonable separation could be obtained for t-PA with a cut-off point of 5.2 ng/ml and for PAI-1 18 ng/ml. The sensitivity and specificity for these cut-off points were for t-PA 73% (95% confidence interval 63%-84%) and for PAI-1 67% (confidence interval 55%-77%). The negative predictive value with a cut-off point t-PA concentration of 5.2 ng/ml was 85% (95% confidence interval 70%-94%). We observed a significantly negative association between concentration of t-PA and fibrinolytic activity (rs = -0.47; p <0.005) and also between PAI-1 and fibrinolytic activity (rs = -0.78; p <0.005).We conclude that a young healthy population is characterized by low protein concentration of t-PA (and PAI-1) compared with young patients with a previous instance of spontaneous vein thrombosis, and we tentatively state that a low protein concentration of t-PA predicts a low risk of spontaneous deep vein thrombosis.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

Influence of Seeding Conditions on Initial Biofilm Development during the Startup of Anaerobic Fluidized Bed Reactors

1989 ◽  
Vol 21 (4-5) ◽  
pp. 157-165 ◽  
Author(s):  
F. Ehlinger ◽  
J. M. Audic ◽  
G. M. Faup
Keyword(s):  
Fluidized Bed ◽  
Future Development ◽  
Protein Concentration ◽  
Specific Activity ◽  
Fluidized Bed Reactor ◽  
Fluidized Bed Reactors ◽  
Support Material ◽  
Biofilm Development ◽  
Initial Biofilm

The characterization of the biofilm of an anaerobic fluidized-bed reactor was completed under standard conditions. The distribution of the fixed protein concentration depended on the level in the reactor. The protein concentration reached 1520 µg.g−1 of support at the top of the reactor and only 1200 µg.g−1 at the bottom after 504 hours of operation but the specific activity of the biofilm was 33×10−4 µM acetate.h−1.mg−1 proteins at the bottom and only 26×10−4 µM.h−1.mg−1 at the top. The efficiency of a fluidized bed reactor and the composition of the biofilm changed with an increase of the pH from 7 to 8.5 during the seeding of the support material. Future development of the biofilm and the specific activity of the support were affected.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica

2019 ◽  
Vol 26 (6) ◽  
pp. 435-448
Author(s):  
Priyanka Biswas ◽  
Dillip K. Sahu ◽  
Kalyanasis Sahu ◽  
Rajat Banerjee
Keyword(s):  
Circular Dichroism ◽  
Steady State ◽  
Entamoeba Histolytica ◽  
Protein Concentration ◽  
Trna Synthetase ◽  
Solvent Exposure ◽  
Steady State Fluorescence ◽  
State Process ◽  
Wide Range ◽  
Circular Dichroïsm

Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

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