Sarcoplasmic and myofibrillar protein fractions were isolated from 2 g of chopped meat according to the procedures of Pietrzak et al. (1997) (link). A biuret assay was performed to determine the protein concentration of the 2 fractions. Samples were diluted to 2 mg/mL in sample buffer (8 mol urea, 2 mol thiourea, 3% SDS (wt/vol), 75 mmol DTT, 25 mmol Tris-HCl (pH 6.8), 0.004% bromophenol blue) and denatured for 3 min in boiling water. Denatured protein samples (15 μg protein/lane) and a broad range molecular weight standard (5 to 250 kDa, Thermo Scientific PageRuler Broad Range Unstained Protein Ladders, Waltham, MA) were loaded onto Novex precast 4–20% tris-glycine polyacrylamide gels (Life Technologies Corp., Carlsbad, CA) and ran at 4°C at a constant voltage. Gradient gels (4–20%) were utilized to allow for a broader range of proteins to be analyzed, and equal protein loads ensured that differences were because of actual variations in the protein profiles. Gels were then stained (Coomassie brilliant blue R-250) and destained. The densities of 13 sarcoplasmic and 17 myofibrillar protein bands were quantified by Alpha View software (v 3.4, ProteinSimple Inc., Santa Clara, CA); the relative abundance of each individual protein band was expressed as a percentage of the total protein abundance of all bands in the lane.
Pageruler unstained broad range protein ladder
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
The PageRuler Unstained Broad Range Protein Ladder is a pre-stained protein standard used for estimating the molecular weight of proteins in protein gel electrophoresis. It contains a mixture of proteins with a wide range of molecular weights, allowing for the determination of protein size in a broad size range.
Automatically generated - may contain errors
Lab products found in correlation
Novex precast 4 20 tris glycine polyacrylamide gels, by Thermo Fisher Scientific (2 mentions)
Sypro ruby, by Thermo Fisher Scientific (2 mentions)
Pac 300 power supply, by Bio-Rad (2 mentions)
Mini protean 2 dual slab cell system, by Bio-Rad (2 mentions)
Coomassie brilliant blue r 250 dye, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Honeywell (1 mentions)
Pure ethanol, by ITW Reagents (1 mentions)
Hydrochloric acid (hcl), by ITW Reagents (1 mentions)
Deionized water, by Merck Group (1 mentions)
Curcumin, by Merck Group (1 mentions)
Quantity one 1 d, by Bio-Rad (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Mini protean tetra system, by Bio-Rad (1 mentions)
Owl dual gel vertical electrophoresis systems, by Thermo Fisher Scientific (1 mentions)
Amersham typhoon bioimager, by GE Healthcare (1 mentions)
Mini protean 2 dual, by Bio-Rad (1 mentions)
Protean 2 xi cell, by Bio-Rad (1 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Coomassie blue g 250, by Merck Group (1 mentions)
17 protocols using pageruler unstained broad range protein ladder
1
SDS-PAGE Analysis of Meat Proteins
2
SDS-PAGE Analysis of Myofibrillar Proteins
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Myofibrillar protein fractions were isolated from 2 g of minced meat according to the procedure of Pietrzak et al. (1997) (link). A biuret assay was performed to determine protein concentrations. Samples were then diluted to a protein concentration of 2 mg/ml in sample buffer (8 mol urea, 2 mol thiourea, 3% SDS (wt/vol), 75 mmol DTT, 25 mmol Tris-HCl (pH 6.8), 0.004% bromophenol blue) and denatured for 3 min in boiling water. Denatured protein samples (15 µg protein/lane) and a broad range molecular weight standard (5–250 kDa, Thermo Scientific PageRuler Broad Range Unstained Protein Ladders, Waltham, MA, United States) were loaded onto Novex precast 4%–20% tris-glycine polyacrylamide gels (Life Technologies Corp., Carlsbad, CA, United States) and ran at 4°C at a constant voltage. Gradient gels were utilized to allow for a broader range of proteins to be analyzed, and equal protein loads ensured that differences were because of actual variations in the protein profiles. Gels were then stained (Coomassie brilliant blue R-250) and destained. The densities of 13 myofibrillar protein bands were quantified using Alpha View software (v 3.4, ProteinSimple Inc., Santa Clara, CA, United States). The relative abundance of each individual protein band was expressed as a percentage of the total protein abundance of all bands within the lane.
3
Phage Protein Separation by SDS-PAGE
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Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed by the method of Laemmli [53 (link)]. A sample of 50 μl purified phage particles (5×1010 pfu/ml) was dissolved in 50 μl loading buffer (50 μl Mercaptoethanol, 950 μl Laemmli sample buffer (2×) for SDS-PAGE; SERVA electrophoresis). After heating at 95°C for 5 min, the samples were subjected to electrophoresis in 12% SDS-PAGE gel along with protein marker (PageRuler Broad Range Unstained protein ladder; Thermo Scientific) with Tris-glycine as running buffer. After electrophoresis, proteins were visualized by staining with Coomassie Brilliant Blue R250 dye (Sigma).
4
Protein Purification and Characterization Protocol
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Lactoferrin-Lf was purchased from DMV International (USA) and Glycomacropeptide-GMP was kindly offered by Davisco Food International, INC. (Le Sueur, USA). To prepare the samples, it was used deionized water purified to a resistance of 15 MU, Millipore Corp. (France).
Hydrochloric acid was purchased from Panreac, Spain and sodium hydroxide was obtained from Riedel-de Haen (Germany). Hydrophilic model compound, caffeine was purchased from VWR (USA) and Amicon ® Ultra-0.5 centrifugal filter of 3 kDa and 8 kDa devices from, Millipore Corp. (Ireland) were used. Lipophilic model compound, curcumin was purchased from SigmaeAldrich, St. Louis and pure ethanol was purchased from Panreac (Barcelona, Spain). Fluorescein isothiocyanate (FITC) was purchased from Fluka (Germany). Standard marker proteins from PageRuler™ Broad Range Unstained Protein Ladder, Lot ##002252 was purchased from Thermo Scientific (Lithuania).
Hydrochloric acid was purchased from Panreac, Spain and sodium hydroxide was obtained from Riedel-de Haen (Germany). Hydrophilic model compound, caffeine was purchased from VWR (USA) and Amicon ® Ultra-0.5 centrifugal filter of 3 kDa and 8 kDa devices from, Millipore Corp. (Ireland) were used. Lipophilic model compound, curcumin was purchased from SigmaeAldrich, St. Louis and pure ethanol was purchased from Panreac (Barcelona, Spain). Fluorescein isothiocyanate (FITC) was purchased from Fluka (Germany). Standard marker proteins from PageRuler™ Broad Range Unstained Protein Ladder, Lot ##002252 was purchased from Thermo Scientific (Lithuania).
5
Quantitative Protein Analysis from Cell Cultures
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The total protein content from culture media and cell fraction was assessed as described previously (Krasauskas et al., 2019 (link)) with some modifications. Briefly, strains were grown stationary in TSB media at 30°C for 30 h. The cells were separated from the media by a centrifugation at 10,000 × g for 10 min at 4°C. The supernatant was further clarified by a filtration through 0.22 μm filter to remove the remaining biomass. The proteins were precipitated by the addition of trichloroacetic acid (TCA) to a final concentration of 10% and centrifuged at 15,000 × g for 60 min at 4°C. The pellet was washed twice with an ice-cold acetone and dried by incubating tube at 95°C. The separated cells and precipitated proteins were then re-suspended with Laemmli-12% SDS-PAGE sample buffer. After electrophoresis, gels were stained with Coomassie brilliant blue. The PageRulerTM unstained broad range protein ladder (5 μl per lane) was used as a marker (Thermo Fisher Scientific).
6
Insect Protein Molecular Weight Analysis
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For determining the molecular weight distribution of the insect proteins SDS-PAGE according to Laemmli (1970) (link) was used. As described by Reinkensmeier et al. (2015) , the pooled samples (n = 3) were mixed in a ratio of 1:10 with sample buffer (0.0125 M Tris buffer at pH 6.8 containing 0.005 M EDTA at pH 6.8–7.0, 1% of sodium dodecyl sulphate, 10% of glycerol, 1% of 2-mercaptoethanol and 0.005% of Bromophenol Blue). Denaturation of the proteins was conducted at 95 °C for 3 min prior to analysis. Vertical electrophoresis equipment (Mini-PROTEAN) from Bio-Rad (Bio-Rad Laboratories GmbH, Munich, Germany) were used to prepare the gels. As standard the PageRulerTM Unstained Broad Range Protein Ladder (Thermo scientific, Vilnius, Lithuania) was used. The band intensity of 5 μl/10 μl of the samples separated in 12% T gels was estimated following staining the gels with Coomassie Brilliant blue and quantification was conducted using analysis Software (Quantity One 1-D, version 4.5.2, Bio-Rad, Milan, Italy).
7
Immunoconjugate Characterization by SDS-PAGE
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Anti-MT1-MMP immunoconjugates were analyzed
using reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Samples were prepared by adding sample buffer (2.5 μL,
NuPAGE 4× LDS sample buffer), dithiothreitol (1 μL, NuPAGE
10× Sample Reducing Agent), and deionized water (5.5 μL)
to each of the antibody samples (1 μL, 1 mg/mL). The resulting
solutions were incubated at 70 °C for 10 min at 450 rpm. Protein
samples and molecular weight standards (ThermoScientific PageRuler
Unstained Broad Range Protein Ladder) were then loaded on to a 10-well
protein gel (4–12% Bis-Tris) and run for 1 h at 200 V in NuPAGE
MOPS SDS running buffer. Once complete, the gel was washed three times
in water (200 mL, 5 min) before staining using Coomassie Fluor Orange
protein stain (50 mL) for 1 h. Destaining was achieved by washing
in acetic acid (1 M) for 5 min before finally washing again in water.
The gel was scanned using a Typhoon Bioimager for Coomassie Fluor
Orange (Cy3, λex = 532 nm) and IRDye800 (Cy7, λex = 785 nm).
using reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Samples were prepared by adding sample buffer (2.5 μL,
NuPAGE 4× LDS sample buffer), dithiothreitol (1 μL, NuPAGE
10× Sample Reducing Agent), and deionized water (5.5 μL)
to each of the antibody samples (1 μL, 1 mg/mL). The resulting
solutions were incubated at 70 °C for 10 min at 450 rpm. Protein
samples and molecular weight standards (ThermoScientific PageRuler
Unstained Broad Range Protein Ladder) were then loaded on to a 10-well
protein gel (4–12% Bis-Tris) and run for 1 h at 200 V in NuPAGE
MOPS SDS running buffer. Once complete, the gel was washed three times
in water (200 mL, 5 min) before staining using Coomassie Fluor Orange
protein stain (50 mL) for 1 h. Destaining was achieved by washing
in acetic acid (1 M) for 5 min before finally washing again in water.
The gel was scanned using a Typhoon Bioimager for Coomassie Fluor
Orange (Cy3, λex = 532 nm) and IRDye800 (Cy7, λex = 785 nm).
8
SDS-PAGE Analysis of Commercial Proteins
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Commercial protein samples have been analyzed by SDS PAGE performed by the Mini-PROTEAN Tetra System (BioRad) with Laemmli gel (10%T/5%C) consisting of separation and comb gel. Samples were prepared in milliQ water at concentration of 3 mg/ml. Samples (20 µl) were mixed with 5 µl of 5x loading buffer (0.3 M Tris-HCl pH = 6.8, 25% β-mercaptoethanol, 50% glycerol, 10% SDS, 1% bromophenol blue), heated to 95 °C for 5 min, and 15 µl of samples were applied to the gel together with 5 µl of “Thermo Scientific PageRuler Unstained Broad Range Protein Ladder”. Electrophoresis was performed with 25 mM Tris, 192 mM Glycine, 0,1% SDS running buffer for about 1 h (130 V). The gel was stained in Coomassie staining solution for overnight followed by destaining in milliQ water and the results, presented in Figure S3 show, that all commercial proteins used have high purity and are suitable for metal-binding studies.
9
Avenin Peptide Polymorphism Analysis by SDS-PAGE
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The polymorphism of avenin peptides was studied in the condition of the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [22 (link)] on 14% separating gels and 4.6% stacking gels in Thermo Scientific™ Owl™ Dual-Gel Vertical Electrophoresis Systems (US) units. Extractable proteins were diluted in the ratio 1:1 (v/v) with the sample buffer (0.055 M Tris–HCl, pH 6.8; 2% SDS; 40% glycerol; 1% DTT; and 0.0025% bromophenol blue) and heated at 90 °C for 5 min. After the run, the proteins were fixed for 30 min in 12% trichloroacetic acid and stained for 30 min with Coomassie Brilliant Blue R-250. Molecular weights of the polypeptides were estimated by using the Thermo Scientific™ PageRuler™ Unstained Broad Range Protein Ladder.
10
SDS-PAGE Analysis of Phage Proteins
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