Tissue lysates containing 30 μg proteins were separated by SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. The membrane was immunoblotted with polyclonal Nrf2 (sc13032, 1:1000), HO-1 (sc1369601:1000), glutamate–cysteine ligase modifier subunit (GCSm, sc22754, 1:1000), GCH-1 (sc376483, 1:1000), and DHFR (sc33184, 1:1000) as primary antibodies, which were purchased from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX), and anti-rabbit IgG conjugated with horseradish peroxidase (31,430, 1:10,000, Thermo Fisher Scientific, Waltham, MA) as a secondary antibody. Primary nNOSα (1:500, ab229785) antibody, α-synuclein (α-S, ab1903, 1:1000), and tyrosine hydroxylase (TH, ab112, 1:200) were purchased from Abcam (Abcam, Cambridge, MA). P-Glycogen synthase kinase-3beta (p-GSK-3β, 9331s, 1:1000) was from Cell Signaling (Cell Signaling Technology, Danvers, MA). Binding of antibodies to the blots was detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer’s instructions. Stripped blots were re-probed with β-actin-specific polyclonal antibodies (sc8432, 1:1000, Santa Cruz Biotechnology, Dallas, TX) to enable normalization of signals between samples. Band intensities were analyzed using Bio-Rad Gel Doc (Bio-Rad, Hercules, CA).
Gel doc
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The Gel Doc is a versatile imaging system designed for the visualization and analysis of DNA, RNA, and protein samples separated by gel electrophoresis. It captures high-quality images of gels and blots, providing researchers with reliable data for their experiments.
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Lab products found in correlation
Image lab software, by Bio-Rad (11 mentions)
Quantity one software, by Bio-Rad (8 mentions)
Image lab, by Bio-Rad (7 mentions)
Gelred, by Biotium (6 mentions)
Sybr gold, by Thermo Fisher Scientific (5 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Nuclear cytosol fractionation kit, by Abcam (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Chef dr 2 system, by Bio-Rad (3 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Horseradish peroxidase, by Bio-Rad (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Quantity one 1 d analysis software, by Bio-Rad (3 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (3 mentions)
337 protocols using gel doc
1
Immunoblotting of Cellular Signaling Proteins
2
Quantitative Cell Transcriptomics Analysis
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• Countess® automated cell counter (Life Technologies, Carlsbad, CA, #10227)
• Countess® Cell Counting Chamber Slide (Life Technologies, #C10228)
• Trypan Blue Stain, 0.04% (Life Technologies, #T10282)
• 72-well Terasaki plates (Fisher Scientific, Pittsburgh, PA, #470378)
• Quick-RNA™ MicroPrep kit (Zymo Research, Irvine, CA, #R1051)
• SYBR Premix Ex Taq™ II (2X) (TaKaRa/Clontech, Mountain View, CA #RR820)
• PCR plates
o e.g., TempPlate No-skirt 0.1 mL PCR plates (USA Scientific, Ocala, FL, #1402-9590)
• Optically-clear PCR film
o e.g., TempPlate RT optically clear film (USA Scientific, Ocala, FL, #2978-2100)
• Gel imaging equipment
o e.g., Bio-Rad Gel Doc (Bio-Rad, Hercules, CA, #170-8170)
• Thermal cycler
o e.g., Veriti® 96-well Thermal Cycler (Life Technologies, #4375786)
• qPCR instrument
o e.g., StepOnePlus™ Real-Time PCR system (Life Technologies, #4376598)
• General lab supplies (microcentrifuge tubes, pipettes, etc.)
• Inverted microscope
o Minimum brightfield (preferred with phase). Optionally with fluorescence capabilities appropriate for the fluorophores applied.
• Countess® Cell Counting Chamber Slide (Life Technologies, #C10228)
• Trypan Blue Stain, 0.04% (Life Technologies, #T10282)
• 72-well Terasaki plates (Fisher Scientific, Pittsburgh, PA, #470378)
• Quick-RNA™ MicroPrep kit (Zymo Research, Irvine, CA, #R1051)
• SYBR Premix Ex Taq™ II (2X) (TaKaRa/Clontech, Mountain View, CA #RR820)
• PCR plates
• Optically-clear PCR film
• Gel imaging equipment
• Thermal cycler
• qPCR instrument
• General lab supplies (microcentrifuge tubes, pipettes, etc.)
• Inverted microscope
3
Cell Fractionation and Western Blot
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Cytoplasmic and nuclear protein extracts were obtained by using the Nuclear/Cytosol Fractionation Kit from Biovision Inc. (Milpitas, CA, USA). Cell extracts and conditioned media from treated and untreated cells were electrophoresed under reducing conditions and transferred to nitrocellulose filter (Schleicher and Schuell GmbH, Dassel, Germany). Reactive bioluminescent bands were visualized by using the detection kit (Supersignal, Perbio Science, Tattenhall, UK) using Bio-Rad gel Doc™ (Bio-Rad Laboratories S.r.l., Milan, Italy).
4
Ubiquitination Assay for Substrate and Chains
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Ubiquitination assays for substrate were performed in PBS buffer (pH 7.4) at 25 °C using 0.25 µM E1 (mouse UBA1), 2 µM E2 (UbcH5b), 0.5 µM E3, 50 µM Ub, 5 mM MgCl2, 5 mM ATP, and 2 µM hGBP1. Reactions were initiated by the addition of ATP. Sampled time points were taken by adding 20 µL of the reaction mixture to 20 µL of reducing SDS-PAGE load dye, followed by boiling prior to electrophoresis. SDS-polyacrylamide gels were loaded with 20 µL of sample, followed by electrophoresis, Coomassie staining, and imaging on a Bio-Rad Gel Doc (Bio-Rad). For immunoblot analysis, SDS-polyacrylamide gels were loaded with 5 µL of sample, followed by electrophoresis, transfer to a PVDF membrane, and incubated with specific primary antibodies as indicated. Ubiquitination assays for the polymerization of free Ub chains were performed as above but without hGBP1 and detected by Coomassie staining. Finally, the signals were visualized by the chemiluminescence system (Perkin Elmer). These experiments were conducted three times. The uncropped and unprocessed western blotting images were show in Supplementary Fig. 11 .
5
Protein Extraction and Immunoblotting from Frozen Liver Samples
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An aliquot of frozen liver samples was homogenized by using (hand held homogenizer) in ice-cold lysis buffer (50 mM Tris-HCL, 150 mNaCl, 1 mM EGTA, 1 mM EDTA, 20 mM Na3 VO4, 0.5% NP-40, 1% Triton X-100, 1 mM PMSF) with protease inhibitor (Protease inhibitor Cocktail Set III, Calbiochem, La Jolla, CA). After homogenization, samples were passed through needle to ensure complete cell lysis and then centrifuged 14,000 rpm for 20 min at 4 °C. The supernatant was immediately used or stored at −80 °C. For immunoblot analysis, 30 µg protein was resolved on 8–12% polyacrylamide gels and transferred to a nitrocellulose membrane. Membranes were incubated with primary antibody at 4 °C for overnight followed with incubation with anti-rabbit or anti-mouse secondary antibody horseradish peroxidase conjugate. All antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Abcam (Cambridge, MA). Blots were developed by using chemiluminescence and autoradiography was done by using Bio-Rad Gel Doc (Bio-Rad laboratories In., Hercules, CA)
6
Quantification of C. elegans Yolk Proteins
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Coomassie staining of worm protein extracts using SDS-PAGE was performed as described in [3 (link),27 (link)]. Synchronous worm populations were grown until day 3 of adulthood, after which 50 animals per sample were picked into 15 μL of M9 buffer. A total of 15 μL of Laemmli sample buffer containing β-mercaptoethanol was added. Samples were incubated for 15 minutes at 70°C, centrifuged at max speed for 5 minutes, and incubated at 95°C for 5 minutes. A total of 15 μL of each sample was loaded on a 4%–12% bis-tris Criterion XT precast polyacrylamide gel (Bio-Rad, Hercules, CA) using XT MOPS as a running buffer. Gels were stained with Coomassie Brilliant Blue and destained with a 40% methanol, 10% acetic acid solution. Gel images were taken with a Bio-Rad Gel Doc (Bio-Rad, Hercules, CA). Identification of YP170, YP115, and YP88 bands is based on [7 (link),27 (link)]. Quantification of yolk protein abundance was done by normalizing to total protein content in a lane using ImageLab 6.0. At least three populations were assayed for each condition. Data were analyzed using a one-way ANOVA with a Tukey post hoc test.
7
PFGE Typing of S. aureus Isolates
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The determination of the clonality of the isolated S. aureus strains was carried out by means of PFGE, the extraction of bacterial DNA and its digestion with the enzyme SmaI was carried out following the methodology previously described [37 (link)]. Samples were run on a CHEF-DR II system (Bio-Rad, USA). Gels were photographed and digitized using a Bio-Rad Gel Doc (Bio-Rad, USA). The band patterns obtained by PFGE were analyzed with Gene Directory and Gene Tools software (Syngene, Cambridge, UK). We applied the unweighted pair group with mathematical average (UPMGA) based on Dice coefficients to obtain the percent similarities. A band position tolerance of 1.25% was established. For strain typing we used the criteria described by Tenover et al. [38 (link)].
8
Sialidase Activity Assay of WSEIV NA
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Recombinant GM2 (100 μM) was incubated with WSEIV NA or BSA at 10 μg/ml. As a positive control, fetuin (100 μM) was incubated with WSEIV NA (10 μg/ml). The reactions were then analyzed using a Sialic Acid (NANA) assay kit (Abcam) as per the manufacturer’s instructions. The amount of free sialic acid was determined by extrapolation from a standard curve and graphed using Graphpad Prism 7. The same samples were also applied using a 700 series microliter syringe (Hamilton) onto pre-scored silica gel TLC plates (Millipore Sigma) and placed in a running buffer of ethanol:acetic acid:water at 5:2:1. As a control for both experiments, GM2 was also incubated with BSA as an irrelevant protein. Assay buffer without GM2 was also applied to the TLC plate as a blank control. The plates were visualized using a Bio-Rad Gel Doc (Bio-Rad), and the chromatogram was annotated on Adobe Illustrator.
9
SDS-PAGE Protein Analysis and Visualization
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Proteins were resolved using a XCell SureLock™ Electrophoresis cell (Invitrogen, Waltham, MA, USA) on 4–12% NuPAGE Bis-Tris gel (Invitrogen) with MOPS SDS running buffer (Invitrogen) at constant 150 V for 1–1.5 h. The SDS-PAGE gel was stained either with Coomassie blue or silver staining procedures.
For Coomassie blue staining, the gel was first fixed in fixing solution (50% methanol and 10% glacial acetic acid) for 1 h with gentle agitation. Thereafter, the gel was stained for 1–2 h with staining solution containing 0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid. Destaining was done by washing the gel in a solution containing 20% methanol and 5% glacial acetic acid. After staining, images were taken using BioRad GelDoc™ (Bio-Rad Laboratories, Copenhagen, Denmark) or Epson perfection V500 photo scanner (Epson, Herlev, Denmark).
The protein gels were blotted on polyvinylidene fluoride (PVDF) membrane, and the zinc binding protein were visualized by dithizone staining, according to Uddin and co-workers [49 (link)].
For Coomassie blue staining, the gel was first fixed in fixing solution (50% methanol and 10% glacial acetic acid) for 1 h with gentle agitation. Thereafter, the gel was stained for 1–2 h with staining solution containing 0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid. Destaining was done by washing the gel in a solution containing 20% methanol and 5% glacial acetic acid. After staining, images were taken using BioRad GelDoc™ (Bio-Rad Laboratories, Copenhagen, Denmark) or Epson perfection V500 photo scanner (Epson, Herlev, Denmark).
The protein gels were blotted on polyvinylidene fluoride (PVDF) membrane, and the zinc binding protein were visualized by dithizone staining, according to Uddin and co-workers [49 (link)].
10
Efficient DNA Assembly Evaluation
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Assemblies were evaluated if the negative controls (either lacking the backbone fragment or one of the insert fragments) had less than a tenth of the number of colonies on the assembly plate. The efficiency of the DNA assembly was calculated by counting the total colony number. The fidelity of assembly for the zeaxanthin pathway plasmid was calculated by dividing the number of yellow colonies by the total colony number. The fidelity of non-zeaxanthin pathway assemblies was assessed by restriction digestion followed by gel electrophoresis, and by DNA sequencing. Plasmid DNA was isolated from resistant clones (Plasmid SV mini, GeneAll Biotechnology, Seoul, Korea), digested with respective enzymes at 37°C for 1 h, electrophoresed in 1% agarose gel at 120V for 20 min and visualized using SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) on a BioRad GEL Doc (BioRad, Hercules, CA, USA) with a XcitaBlue Conversion Screen (BioRad, Hercules, CA, USA). All junction regions of correctly digested constructs were sequenced for junction errors by Sanger sequencing (first BASE, Singapore). For all DNA assemblies performed in this work, the plasmid backbone, which included the selection marker and replication origin in E. coli, was counted as one DNA fragment.
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