FPCount protocol - Short protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Quantification ◽  
Short Version ◽  
Protein Storage ◽  
Concentration Determination ◽  
Sds Page ◽  
Plate Reader ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This short version uses the ECmax protein quantification protocol, and is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the complete protocol, which describes three protein quantification methods. The short protocol also skips the SDS-PAGE steps. If you require these, please see the complete protocol. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 6. Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

FPCount protocol - in-lysate (purification free) protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Quantification ◽  
Protein Storage ◽  
Cell Lysates ◽  
Concentration Determination ◽  
His Tag ◽  
Plate Reader ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression and production of cell lysates. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This in-lysate version of the protocol uses the ECmax protein quantification protocol of FPs in lysates and does not require His-tag purification of the FPs. Note that it is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the 'short' protocol, which requires FP purification, or the 'complete' protocol, which requires FP purification and compares three protein quantification methods. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

FPCount protocol - Full protocol v1

2021 ◽  
Author(s):  
Eszter Csibra ◽  
Guy-Bart Stan
Keyword(s):  
Protein Concentration ◽  
Fluorescent Protein ◽  
R Package ◽  
Protein Storage ◽  
Concentration Determination ◽  
Plate Reader ◽  
Full Protocol ◽  
Microplate Reader

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 5. Gel1: Verification of Expression/Fractions 6. Purification 7. Gel2: Verification of Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 11. Quantification of FP concentration (part2) 12. Protein Storage 13. Calibration of Plate Reader

scholarly journals Quality Attributes of Ultra-High Temperature-Treated Model Beverages Prepared with Faba Bean Protein Concentrates

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1244
Author(s):  
Malik Adil Nawaz ◽  
Tanoj Kumar Singh ◽  
Regine Stockmann ◽  
Hema Jegasothy ◽  
Roman Buckow
Keyword(s):  
Physicochemical Properties ◽  
Faba Bean ◽  
Protein Concentration ◽  
High Concentration ◽  
Molecular Weights ◽  
Oil In Water ◽  
Sds Page ◽  
Lower Protein ◽  
7S Globulin ◽  
Bean Protein

The objective of this research was to develop a model faba bean drink with a high concentration of protein (>4% w/w). The protein molecular weights and frequency for both faba and soy were assessed using SDS-PAGE. Results showed similarities in the protein molecular weight of both faba and soy (mainly 11S globulin ~Glycinin and 7S globulin ~β-conglycinin). Thus, faba can be considered as a potential soy replica in plant-based milk beverages. Oil-in-water emulsions (5–8% w/w available protein) were prepared using faba bean protein concentrate (FPC), 1% sunflower oil, and 0.2% sunflower lecithin. These emulsions were used as model beverages and were further investigated for UHT processibility, stability, and physicochemical properties. The physicochemical properties of emulsions at various processing stages viz., coarse emulsification, homogenisation, and UHT, were measured. An increase in the protein concentration and thermal treatment resulted in an increased oil droplet size, coalescence and flocculation, and protein aggregation. Lower protein concentrations viz., 5–6%, showed greater negative ζ-potential, and thereby, high dispersibility through enhanced electrostatic repulsions than those of higher concentrations (7–8%). Furthermore, an increase in protein concentration and UHT treatment resulted in an increased creaming index. In total, 21 different volatile compounds were detected and quantified, representing different chemical classes, namely alcohols, aldehydes, ketones, esters, furan, and acids. These volatiles have major consequences for the overall flavour chemistry of the model beverage product. Overall, this study showed the potential for application of faba bean as a protein source in UHT-treated legume-based beverages and identified areas for further development.

Changes in casein composition of goats' milk during the course of lactation: physiological inferences and technological implications

1995 ◽  
Vol 62 (3) ◽  
pp. 431-439 ◽  
Author(s):  
Joanna R. Brown ◽  
Andrew J. R. Law ◽  
Christopher H. Knight
Keyword(s):  
Cation Exchange ◽  
Protein Concentration ◽  
Stage Of Lactation ◽  
Sds Page ◽  
Lower Protein ◽  
Kjeldahl Method ◽  
Compositional Changes ◽  
The Individual ◽  
Cheese Production ◽  
Peak Lactation

SummaryFive British Saanen goats were milk sampled during the first 39 weeks of lactation to determine changes in casein composition. Caseins were separated by anion- and cation-exchange FPLC to determine the relative amounts of the individual caseins. Acid, alkaline and SDS-PAGE were used to determine possible genetic polymorphisms and observe any lactational changes. Total casein nitrogen was determined using a micro-Kjeldahl method and this allowed the concentrations of individual caseins to be calculated. The milk of one animal, which had the deduced genotype αs1-CnAB, showed higher concentrations of both total and αs1-casein. The remainder of the group were either heterozygous αs1-CnBE or, more probably, homozygous αs1-CnE and produced milk of a generally lower protein concentration. Both FPLC and PAGE results showed that the relative amounts and concentrations of αs2-casein decreased with stage of lactation, consistent with its susceptibility to proteolysis. The relative amounts of the breakdown products of plasmin attack on β-casein, γ-caseins, were highly negatively correlated with milk yield (r = –0·942, P < 0·001) in the declining phase of lactation, reflecting the gradual involution of the gland at this time. The relative amount of κ-casein increased by ∼ 50% after peak lactation and its concentration almost doubled near the end of lactation. These compositional changes may alter the processing qualities of goats' milk in relation to cheese production.

scholarly journals Heat and Pressure Treatments on Almond Protein Stability and Change in Immunoreactivity after Simulated Human Digestion

Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1679 ◽  
Author(s):  
Elisabetta De Angelis ◽  
Simona Bavaro ◽  
Graziana Forte ◽  
Rosa Pilolli ◽  
Linda Monaci
Keyword(s):  
Protein Stability ◽  
Total Protein ◽  
Protein Pattern ◽  
Protein Quantification ◽  
Protein Profiling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Total Protein Content ◽  
Bioinformatic Tools ◽  
Sds Page ◽  
Allergenic Proteins

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

Urine protein quantification in stacking gel by SDS-PAGE

2018 ◽  
Vol 40 (4) ◽  
pp. 487-490
Author(s):  
Jia Jia ◽  
Jie Pan ◽  
Hongpan Xu ◽  
Sen Wang ◽  
Bing Bai
Keyword(s):  
Protein Quantification ◽  
Urine Protein ◽  
Sds Page

scholarly journals Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anand Chopra ◽  
William G. Willmore ◽  
Kyle K. Biggar
Keyword(s):  
Light Exposure ◽  
Covalent Modification ◽  
Protein Quantification ◽  
Visible Range ◽  
Uv Absorbance ◽  
Polyacrylamide Gels ◽  
Fluorescent Emission ◽  
Tyrosine Residues ◽  
Sds Page ◽  
Low Volume

Abstract The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450 nm, with a 310 nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280 nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.

Targeting and processing of membrane-anchored YFP fusion proteins to protein storage vacuoles in transgenic tobacco seeds

2005 ◽  
Vol 15 (4) ◽  
pp. 361-364 ◽  
Author(s):  
Ka Leung Fung ◽  
Yin Fun Yim ◽  
Yu Chung Tse ◽  
Yansong Miao ◽  
Samuel S.M. Sun ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Fusion Proteins ◽  
Seed Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Protein Storage ◽  
Protein Storage Vacuoles ◽  
Pharmaceutical Proteins ◽  
Tobacco Seeds ◽  
Foreign Proteins

Seeds that store proteins in protein storage vacuoles are attractive bioreactors for producing and storing large amounts of pharmaceutical proteins. However, foreign proteins expressed in transgenic plants are subjected to the delivery and modification processes present within plant cells. Here, it is demonstrated that unique membrane sequences deliver a yellow fluorescent protein (YFP) to the seed protein storage vacuoles in transgenic tobacco (Nicotiana tabacum L.) plants, where the YFP is then separated from its membrane anchors. This precise targeting and separation is required for the successful delivery of useful proteins to seed protein storage vacuoles for their stable accumulation in transgenic crops.

Thermal gelation and denaturation of bovine β-lactoglobulins A and B

1994 ◽  
Vol 61 (2) ◽  
pp. 221-232 ◽  
Author(s):  
Mary McSwiney ◽  
Harjinder Singh ◽  
Osvaldo Campanella ◽  
Lawrence K. Creamer
Keyword(s):  
Heat Treatment ◽  
Rheological Properties ◽  
Functional Property ◽  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Gel Formation ◽  
Native Page ◽  
Thermal Gelation ◽  
Sds Page ◽  
Important Functional Property

SummaryHeat-induced gelation, an important functional property of β-lactoglobulin, was studied by measuring the rheological properties of both the A and B variants of the protein during and after heat treatment within a range of pH, temperature and concentration. Gel electrophoresis was used to determine the extent of denaturation and disulphide bond crosslinking of some samples. Both variants formed gel networks on heating at temperatures > 75 °C, and under most conditions the storage modulus (G′) of βlactoglobulin A gels was higher than the G′ of β-lactoglobulin B gels, in particular after cooling to 25 °C. A minimum protein concentration of 50 g/1 was required for gel formation at pH 7·0 in 0·1 M-NaCl by both variants at 80 °C. Increasing the protein concentration above 50 g/1 increased G′, the extent of increase being much greater for the A variant than the B variant. G′ of variant A gels was not much influenced by pH whereas G′ of variant B gels decreased slightly from pH 3 to pH 6 and increased between pH 6 and pH 9. When mixtures of the two variants were gelled G′ increased at the temperature of heating (80 °C) and after cooling (25 °C) as the relative quantity of variant A was increased. Comparisons of the loss of discrete protein bands from electrophoretic gels (native-PAGE, SDS-PAGE and SDS-PAGE of reduced samples) showed that heating β-lactoglobulin solutions of 100 g/1 at pH 7 in 0·1 M-NaCl and at 75, 80 and 85 °C caused a faster loss of both native and SDS-soluble β-lactoglobulin A than of β-lactoglobulin B. It was concluded that the loss of native β-lactoglobulin structure from these solutions during heating was faster than the formation of disulphidelinked aggregates, which was faster than gel formation for both β-lactoglobulin A and β-lactoglobulin B, and that each of these reactions was faster for β-lactoglobulin A than for β-lactoglobulin B. This contrasts with conclusions drawn from some previous studies and may arise from the differences in protein concentration between the present study (∼ 100 g/1) and the previous ones (< ∼ 10 g/1).

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