scholarly journals Comparative evaluation of multiple protein extraction procedures from three species of the genus Caulerpa

2021 ◽  
Author(s):  
Sarah Caronni ◽  
Filippa Addis ◽  
Maria Anna Delaria ◽  
Rodolfo Gentili ◽  
Chiara Montagnani ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Yield ◽  
High Yield ◽  
Extraction Procedures ◽  
Multiple Protein ◽  
Biomass Ratio ◽  
Pre Treatment ◽  
Set Up

AbstractThe aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus Caulerpa (the invasive C. taxifolia and C. cylindracea and the autochthonous C. prolifera). Five multi-step protein extraction procedures, set up for other macroalgal species, were tested. For each of them, different pre-treatment and extraction conditions were simultaneously examined, according to a factorial design, considering the starting material, the solvent-to-biomass ratio, and the incubation temperature. Protein yield in the obtained extracts was estimated with the Bradford method. Further, polyacrylamide gel electrophoresis (SDS-PAGE) was used to resolve proteins, assessing their quality and integrity. Significant differences in protein yield were observed among the extraction protocols and the conditions tested, also in relation to the considered species. Profiles having an acceptable quality were obtained for C. prolifera and C. cylindracea, and from the obtained results, the best method to obtain high yield and quality protein extracts for the two above-mentioned species appears to require the use of a primary TCA/acetone extraction buffer followed by a lysis buffer with NaCl, KCl, urea, Triton, SDS and a protease inhibitor. The best results, in particular, were obtained starting from fresh pulped material with a buffer-to-biomass ratio of 10:1 and an incubation temperature of 4°C. For C. taxifolia, instead, none of the tested protocols produced satisfactory results and further studies will be required.

scholarly journals Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

2020 ◽  
Author(s):  
Dominic Esposito ◽  
Jennifer Mehalko ◽  
Matthew Drew ◽  
Kelly Snead ◽  
Vanessa Wall ◽  
...  
Keyword(s):  
Protein Production ◽  
Incubation Temperature ◽  
Recombinant Protein Expression ◽  
Mammalian Cell Culture ◽  
Spike Protein ◽  
Hek293 Cells ◽  
High Yield ◽  
Limiting Factor ◽  
Yield Production ◽  
Multiple Protein

The SARS-CoV-2 spike trimer is the primary antigen for several serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Until stable cell lines are developed to increase the titer of this secreted protein in mammalian cell culture, the low yield of spike protein produced from transient transfection of HEK293 cells will be a limiting factor for these assays. To improve the yield of spike protein and support the high demand for antigens in serology assays, we investigated several recombinant protein expression variables by altering the incubation temperature, harvest time, chromatography strategy, and final protein manipulation. Through this investigation, we developed a simplified and robust purification strategy that consistently yields 5 mg of protein per liter of expression culture for two commonly used forms of the SARS-CoV-2 spike protein. We show that these proteins form well-behaved stable trimers and are consistently functional in serology assays across multiple protein production lots.

scholarly journals A comparative study on the extraction effects of common agents on collagen-based binders in mural paintings

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Jianghao Du ◽  
Zhanyun Zhu ◽  
Junchang Yang ◽  
Jia Wang ◽  
Xiaotong Jiang
Keyword(s):  
Protein Structure ◽  
Comparative Study ◽  
Protein Extraction ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Process ◽  
Ultraviolet Absorption Spectrum ◽  
Mural Paintings ◽  
The Impact

AbstractIn this paper, a comparative study was conducted on the extraction effects of six agents for collagen-based mural painting binders. These agents were used to extract the residual proteins in the non-aged and thermal aged samples. The protein extraction efficiencies of different extracting agents were quantitatively determined by bicinchoninic acid (BCA) method, and then processed by multivariate analysis of variance (MANOVA). The impact of the extraction process on the protein structure was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet absorption spectrum (UV) and circular dichroism (CD). The results showed that, for both non-aged and aged samples, the extraction efficiency of 2 M guanidine hydrochloride (GuHCl) was significantly higher than the other five agents, with less damage to the protein structure during the extraction process.

scholarly journals A Single Seed Protein Extraction Protocol for Characterizing Brassica Seed Storage Proteins

Agronomy ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 107
Author(s):  
Mahmudur Rahman ◽  
Lei Liu ◽  
Bronwyn J. Barkla
Keyword(s):  
Seed Protein ◽  
Storage Proteins ◽  
Protein Extraction ◽  
Seed Storage ◽  
Seed Storage Proteins ◽  
Protein Yield ◽  
Tissue Type ◽  
Single Seed ◽  
Material Optimization ◽  
Hazardous Chemical

Rapeseed oil-extracted expeller cake mostly contains protein. Various approaches have been used to isolate, detect and measure proteins in rapeseeds, with a particular focus on seed storage proteins (SSPs). To maximize the protein yield and minimize hazardous chemical use, isolation costs and the loss of seed material, optimization of the extraction method is pivotal. For some studies, it is also necessary to minimize or avoid seed-to-seed cross-contamination for phenotyping and single-tissue type analysis to know the exact amount of any bioactive component in a single seed, rather than a mixture of multiple seeds. However, a simple and robust method for single rapeseed seed protein extraction (SRPE) is unavailable. To establish a strategy for optimizing SRPE for downstream gel-based protein analysis, yielding the highest amount of SSPs in the most economical and rapid way, a variety of different approaches were tested, including variations to the seed pulverization steps, changes to the compositions of solvents and reagents and adjustments to the protein recovery steps. Following SRPE, 1D-SDS-PAGE was used to assess the quality and amount of proteins extracted. A standardized SRPE procedure was developed and then tested for yield and reproducibility. The highest protein yield and quality were obtained using a ball grinder with stainless steel beads in Safe-Lock microcentrifuge tubes with methanol as the solvent, providing a highly efficient, economic and effective method. The usefulness of this SRPE was validated by applying the procedure to extract protein from different Brassica oilseeds and for screening an ethyl methane sulfonate (EMS) mutant population of Brassica rapa R-0-18. The outcomes provide useful methodology for identifying and characterizing the SSPs in the SRPE.

scholarly journals Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jack Jingyuan Zheng ◽  
Joanne K. Agus ◽  
Brian V. Hong ◽  
Xinyu Tang ◽  
Christopher H. Rhodes ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cholesterol Acyltransferase ◽  
Density Lipoprotein ◽  
Size Exclusion ◽  
High Yield ◽  
Phospholipid Transfer ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Alpha 1 Antitrypsin

AbstractHigh-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

scholarly journals UVA-LED Technology’s Treatment Efficiency and Cost in a Competitive Trial Applied to the Photo-Fenton Treatment of Landfill Leachate

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 1026
Author(s):  
Javier Tejera ◽  
Antonio Gascó ◽  
Daphne Hermosilla ◽  
Víctor Alonso-Gomez ◽  
Carlos Negro ◽  
...  
Keyword(s):  
Landfill Leachate ◽  
Mercury Vapor ◽  
Cod Removal ◽  
Treatment Efficiency ◽  
Alternative Source ◽  
Effluent Quality ◽  
Radiation Sources ◽  
Initial Ph ◽  
Pre Treatment ◽  
Set Up

The objective of this trial was to assess the application of UVA-LED technology as an alternative source of irradiation for photo-Fenton processes, aiming to reduce treatment costs and provide a feasible treatment for landfill leachate. An optimized combination of coagulation with ferric chloride followed by photo-Fenton treatment of landfill leachate was optimized. Three different radiation sources were tested, namely, two conventional high-pressure mercury-vapor immersion lamps (100 W and 450 W) and a custom-designed 8 W 365 nm UVA-LED lamp. The proposed treatment combination resulted in very efficient degradation of landfill leachate (COD removal = 90%). The coagulation pre-treatment removed about 70% of the COD and provided the necessary amount of iron for the subsequent photo-Fenton treatment, and it further favored this process by acidifying the solution to an optimum initial pH of 2.8. The 90% removal of color improved the penetration of radiation into the medium and by extension improved treatment efficiency. The faster the Fenton reactions were, as determined by the stoichiometric optimum set-up reaction condition of [H2O2]0/COD0 = 2.125, the better were the treatment results in terms of COD removal and biodegradability enhancement because the chances to scavenge oxidant agents were limited. The 100 W lamp was the least efficient one in terms of final effluent quality and operational cost figures. UVA-LED technology, assessed as the application of an 8 W 365 nm lamp, provided competitive results in terms of COD removal, biodegradability enhancement, and operational costs (35–55%) when compared to the performance of the 450 W conventional lamp.

Studies on the use of cultured cells in a bioassay for parathyroid hormone

1994 ◽  
Vol 143 (2) ◽  
pp. 261-268
Author(s):  
A E Armston ◽  
P J Wood
Keyword(s):  
Parathyroid Hormone ◽  
Cyclic Amp ◽  
Cell Line ◽  
Cultured Cells ◽  
Biologically Active ◽  
Kidney Cell Line ◽  
Opossum Kidney ◽  
Pre Treatment ◽  
Set Up ◽  
Insufficient Sensitivity

Abstract Measurement of parathyroid hormone (PTH) is important for diagnosing hyper- and hypoparathyroidism. The move to two-site immunometric assays that detect the whole molecule has improved the discrimination of these conditions but these assays may be too restrictive because some PTH fragments that are biologically active may not be detected. In addition, PTH-like peptide of malignancy, an important cause of malignancy-associated hypercalcaemia, is not detected by the two-site assays. Experiments were performed to set up a simple, robust and inexpensive bioassay for PTH, exploiting a kidney cell line and using cyclic AMP or an eluted stain assay as the end point. Of the 12 cell lines tested, an opossum kidney (WOK) cell line showed the most promise. Despite optimization of the procedure to include pre-treatment with dexamethasone, insulin and PTH, followed by incubation in the presence of 5′ -guanylimidodiphosphate, isobutyl-1-methylxanthine and forskolin, the WOK cells showed insufficient sensitivity for use in a cultured cell bioassay for PTH in human serum. In addition, the cells were less sensitive to PTH-like peptide precluding their use for an assay for this molecule. Journal of Endocrinology (1994) 143, 261–268

scholarly journals Evaluation of effective protein extraction procedure to profile petiole of Moringa oleifera

2020 ◽  
Vol 7 (2) ◽  
pp. 214
Author(s):  
Zetty Amirah Zulkifli ◽  
Zaidah Rahmat
Keyword(s):  
Moringa Oleifera ◽  
Protein Extraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extraction Procedure ◽  
Electrophoretic Pattern ◽  
Antioxidant Property ◽  
Extraction Methods ◽  
Protein Profiling ◽  
Sds Page

Moringa oleifera is widely known as multipurpose tree since all of its parts confer multiple functions. The leaf is highly favourable among consumers while the petiole is mostly wasted. There are numerous studies on the flavonoid and antioxidant property of the stem and twig. However, study on the petiole has never been done. There-upon, this study was conducted to develop protein profiling of the petiole. In this study, 6 different protein extraction methods were tested on the fresh petiole before its protein quantity and quality were checked via Bradford assay and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) respectively. The in-solution digestion was then done prior to LC-MS/MS analysis. The protein electrophoretic pattern from the SDS-PAGE proves that method 6 using Tris HCl buffer with incorporation of dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) confers the best quality of protein. It produced the highest number of visible individual bands compared to other methods. Meanwhile, 93 proteins were successfully identified via LCMS analysis where the protein, signal response and carbohydrate metabolism categories confer the highest percentage. High quality and content of the protein extracted from the petiole including the antioxidant, anticancer and antidiabetic protein identified suggested that consuming this part of the plant could enhance nutrients of human body.

scholarly journals Practical guidance in the use of image-guided radiotherapy systems

2016 ◽  
Vol 5 (1) ◽  
Author(s):  
A. Pastorino ◽  
L. Todisco ◽  
E. Cazzulo ◽  
L. Berretta ◽  
A. Orecchia ◽  
...  
Keyword(s):  
Electronic System ◽  
Image Guided Radiotherapy ◽  
Image Guided ◽  
Pre Treatment ◽  
Portal Images ◽  
Set Up ◽  
Geographic Miss ◽  
Technical Solutions ◽  
Modern Image ◽  
Delivered Dose

From mega-voltage portal images acquired on an electronic system (EPID), technological research has developed 3D and recently 4D volumetric verification modalities, allowing a direct visualization of the target, a direct comparison with the planning-TC and an optimization of the treatment (reduction of set-up errors, verification of the need for re-planning), leading to the very modern Image Guided RadioTherapy (IGRT). IGRT allows different technical solutions through direct or indirect visualization of the tumor and the acquisition of pre-treatment verification images, allowing to identify, quantify and correct errors related to set-up and organ-tumor motion, obtaining a greater compliance of the delivered dose, decreasing the risk of "geographic miss" and toxicity to healthy tissues and reducing the margins from CTV to PTV for the implementation of "dose escalation" protocols.

scholarly journals Characterization of Collagen from Sakhalin Taimen Skin as Useful Biomass

2020 ◽  
Vol 58 (4) ◽  
Author(s):  
Takeshi Nagai ◽  
Masataka Saito ◽  
Yasuhiro Tanoue ◽  
Norihisa Kai ◽  
Nobutaka Suzuki
Keyword(s):  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
High Yield ◽  
Collagen Molecule ◽  
Edible Portion ◽  
Sakhalin Taimen ◽  
Skin Collagen ◽  
Modification Technique ◽  
Cold Acetone ◽  
Α Helix

Research background. Animal collagen has been widely utilized in foods, cosmetics, and biomedical fields. The non-edible portion, such as fish skins and bones, are generated during cooking processes. Most of them are currently discarded as wastes, although the nutritional values of the skins and bones are high. It needs to utilize the non-edible portion for the reduction of environmental impact, as it may be one of source of environmental pollution. Experimental approach. Collagen was prepared from Sakhalin taimen skins as wastes generated during cooking processes. Next, the color, SDS-polyacrylamide gel electrophoresis, ultraviolet absorption, subunit composition, amino acid composition, denaturation temperature, and attenuated total reflectance-Fourier transform infrared spectroscopy analysis were conducted to explore the properties of the collagen. Lastly, it tried to improve the functional properties of the collagen using chemical modification technique for future applications. Results and conclusions. Cold acetone treatment made it possible to easily remove the fats and pigments from skins. The odorless and pure-white collagen was obtained with high-yield. The α3 chain did not exist in the collagen. Sakhalin taimen skin collagen had rich α-helix and low β-sheet structures. Succinylation caused the secondary structural changes of the collagen molecule. Moreover, succinylation made it possible not only to increase the viscosity of collagen solution and but also to improve the solubility of collagen in the physiological conditions around pH=6. Novelty and scientific contribution. This finding was the first report on the absence of the α3 chain in Salmonid fish skin collagens. The succinylated collagen from Sakhalin taimen skins as useful biomass has potential to utilize in foods, cosmetics, and its related industries.

Temperature as a key factor for successful inoculation of soybean with Bradyrhizobium spp. under cool growing conditions in Belgium

2018 ◽  
Vol 156 (4) ◽  
pp. 493-503 ◽  
Author(s):  
J. Pannecoucque ◽  
S. Goormachtigh ◽  
J. Ceusters ◽  
J. Debode ◽  
C. Van Waes ◽  
...  
Keyword(s):  
Field Trials ◽  
Protein Yield ◽  
Control Treatment ◽  
Rhizobial Symbiosis ◽  
Key Factor ◽  
Growing Conditions ◽  
Set Up ◽  
Biological Nitrogen ◽  
Intermediate Performance

AbstractBacterial inoculation of soybean seeds to improve biological nitrogen fixation is a well-known practice to achieve higher seed and protein yield with reduced fertilization. The optimal inoculation strategy in temperate regions is unknown because soybeans are rarely cultivated under colder growing conditions. The aim of the present work was to determine the most suitable inoculation strategy for soybean cultivation in Belgium. Field trials were set up with four Bradyrhizobium inoculants (HiStick, Force 48, Biodoz and Optimize) at two locations over 2 years (2014–2015) and compared with a non-inoculated control treatment. In addition, HiStick was tested at three doses and Optimize at two time periods prior to sowing. Under Belgian conditions, all inoculants were effective in establishing rhizobial symbiosis, resulting in increased yield, protein content, protein yield and thousand-grain weight compared with the non-inoculated control. A single dose of HiStick was sufficient to establish symbiosis. Pre-inoculation with Optimize 2 weeks before sowing gave an intermediate performance for most parameters between the non-inoculated control treatment and inoculation with Optimize 24 h prior to sowing. Among the four products tested, Biodoz seemed the best product for inoculation under cool growing conditions. Based on the atpD gene, the bacterial strain of Biodoz showed complete similarity with Bradyrhizobium diazoefficiens, while strains of other products were identified as Bradyrhizobium japonicum. In vitro growing capacity of the Biodoz strain at 8 °C was higher compared with the other strains. Better cold adaptation of the Biodoz strain might be a possible explanation for the better performance of Biodoz in Belgium.

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