Samples were prepared by adding loading buffer (Bio-Rad) to samples (2:1) and heating at 95°C for 5 min. If denaturation was required, loading buffer + 5% β-mercaptoethanol was used. 10-μL samples including Novex Sharp Pre-stained Protein Standard were loaded onto Bis-Tris 10% ready-made gels (both Invitrogen). The gel was run in an XCell SureLock Mini-Cell (Invitrogen) at 200 V for 1 hr in MOPS buffer (50 mM 3-(N-morpholino)propanesulfonic acid, 50 mM Tris Base, 0.1% SDS, and 1 mM EDTA [pH 7.7]). Gels were stained with EZBlue according to the manufacturer’s instructions to visualize bands. Images of gels were taken with a Hewlett Packard (Newcastle, UK) scanner.
Sharp pre stained protein standard
Manufactured by Thermo Fisher Scientific
The Sharp Pre-stained Protein Standard is a molecular weight marker used to estimate the molecular weights of proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) experiments. It consists of a mixture of pre-stained proteins with known molecular weights, which can be easily visualized during the electrophoresis process.
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Lab products found in correlation
Loading buffer, by Bio-Rad (1 mentions)
Xcell surelock mini cell, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent peroxidase substrate, by Cytiva (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Gradient sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Thermal cycler, by PerkinElmer (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Tris glycine sds buffer, by Bio-Rad (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Any kd mini protean tgx stain free protein gel, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Tricine sample buffer, by Bio-Rad (1 mentions)
Cdp star chemiluminescent substrate, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Submarine system, by Thermo Fisher Scientific (1 mentions)
Mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Seeblue plus2 protein standard, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions)
8 protocols using sharp pre stained protein standard
1
SDS-PAGE Protein Separation Protocol
2
Western Blot Analysis of HIF-1α and HIF-2α
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For western blot analysis, cell lysates were separated on 4–12% SDS polyacrylamide gradient gels (Invitrogen). Proteins were blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). HIF-1α protein was detected using a HIF-1α specific monoclonal antibody (BD Transduction Laboratories) at a dilution of 1∶250. HIF-2α protein was detected using a HIF-2α specific polyclonal antibody (Novus Biologicals) at a dilution of 1∶1000. Anti-β-actin antibody served as a loading control. Binding of the antibodies was visualized by binding of a horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Pharmacia Biotech), and subsequently enhanced using chemiluminescence (Chemiluminescent Peroxidase Substrate), according to the manufactureŕs instructions. Novex Sharp Pre-stained Protein Standard (Invitrogen) was used as molecular weight marker.
3
Protein Hydrolysis Profiling of Hoki Roe
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One dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used to display the protein hydrolysis profiles of hydrolysed hoki roe homogenate protein, using Bolt gradient (4–12 %) Bis-Tris gels (Ryder et al., 2015 (link)). An aliquot (20 µL) of a protein containing sample was added to 7.6 µL Bolt LDS sample buffer containing lithium dodecyl sulphate (LDS) at pH 9.5 and 3.0 µL Bolt™ sample reducing agent containing 500 mM dithiothreitol (DTT). All Bolt reagents were purchased from Invitrogen Life Technologies, Thermo Scientific, Auckland, New Zealand. A programmable Thermal Cycler (Perkin-Elmer, USA) was used to incubate the samples for 5 min at 70 °C, then cooled before loading in gel lanes. Novex Sharp Pre-Stained protein standard (Invitrogen) was loaded in one lane. The electrophoresis was carried out at room temperature in 1 × Bolt MES SDS running buffer for 35 min at 165 V. After electrophoresis, the gel was washed with milli-Q Type 1 water four times, each for 10 min, and then stained overnight with gentle shaking in 20 mL SimplyBlue SafeStain (Invitrogen) followed by destaining in water, and an image was captured with a Canon CanoScan LiDE 600F scanner.
4
Snake Venom Protein Separation
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The proteins from snake venom (3.3 µg) were separated by a 4%–12% Bis-Tris Plus Gel electrophoresis system. The samples were prepared under non-reducing conditions and electrophoresed at a constant voltage according to the manufacturer’s instructions. The protein molecular weight marker (Novex® Sharp Pre-stained Protein Standard, 3.5–260 kDa) was loaded at the same volume as the samples in every gel. After electrophoresis, the gels were soaked in 0.1% Coomassie Brilliant Blue solution containing 50% methanol and 10% acetic acid and shaken for 1 h at room temperature. Subsequently, the staining solution was replaced by destaining buffer (40% methanol and 10% acetic acid) to remove the residual dye until the gel became transparent again. All experiments were repeated at least three times.
5
SDS-PAGE Analysis of Protein Purification
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Samples for SDS–PAGE analysis of the purification progress were prepared using 2× Laemmli sample buffer (Bio-Rad). Samples for analysis via SDS–PAGE followed by western immunoblotting were loaded onto a 4–12% Bis–Tris NuPAGE gel (Novex), along with Sharp Pre-stained protein standard (Novex) and run at 200V in 1× MES SDS running buffer (NuPAGE). Samples for SDS–PAGE analysis were loaded onto an any-kD Mini Protean TGX Stain-free protein gel (Bio-Rad), along with Precision plus unstained marker (Bio-Rad) and run at 300 V in 1× Tris/Glycine/SDS buffer (Bio-Rad). any-kD Mini Protean TGX Stain-free protein gels were visualized using the Chemidoc MP system (Bio-Rad). The loaded sample volume ranged between 15 and 20 µl.
6
SPA-tagged Protein Detection Protocol
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The SPA-tagged strains were grown in the respective growth medium used in ribosome profiling experiments, and harvested. Whole cells were resuspended in tricine sample buffer (Bio-Rad, Hercules, CA) and heated at 95° for 10 min. The total protein (equivalent to the number of cells at OD600 0.05) was separated on a 16.5% tricine gel (Bio-Rad) and transferred to a PVDF membrane (Bio-Rad) according to the manufacturer’s protocol. The SPA-tagged protein was detected using a monoclonal anti-FLAG M2-alkaline phosphatase-conjugated antibody (Sigma-Aldrich, Saint Louis, MO) and CDP Star chemiluminescent substrate (Sigma-Aldrich) according to the manufacturer’s protocol. The Novex sharp prestained protein standard (Novex, Carlsbad, CA) was used as a size marker.
7
SDS-PAGE, Protein Transfer, and Immunostaining
8
Immunoblot analysis of BDBV GP
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Aliquots of thermolysin-treated or mock-treated purified virions were heated for 10 min at 95ºC and separated in Nu-PAGE 4 to 12% Bis-Tris gel with Novex Sharp Pre-Stained Protein Standard used as a molecular weight marker. Proteins were transferred to a nitrocellulose membrane using the iBlot Gel transfer system (Life Technologies). The membrane was incubated with primary rabbit polyclonal antibodies against BDBV GP (1:500; IBT Bioservices) and secondary goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (1:500; KPL). Protein bands were visualized using the chromogenic 4CN two-component peroxidase substrate system (KPL).
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