Protein extraction, separation by electrophoresis, transfer to membranes, membrane incubation with antibodies and washes, and chemiluminescence detection were carried out as described (22 (link)). An anti-Myc tag polyclonal antibody (PA1-981; Thermo Fisher Scientific) and goat horseradish peroxidase–conjugated anti-rabbit IgG (H + L) (Thermo Fisher Scientific) were diluted at 1:2,000 and 1:10,000 and used as primary and secondary antibodies, respectively. The intensities of the bands corresponding to the Myc-tagged fusion proteins were quantified using ImageQuant TL software (GE Healthcare, Chicago, IL). These intensities were then utilized to normalize the relative expression levels of the PhERF1-regulated metabolic genes, which were determined through RT-qPCR analysis.
Goat horseradish peroxidase conjugated anti rabbit igg h l
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat horseradish peroxidase–conjugated anti-rabbit IgG (H + L) is a secondary antibody that binds to rabbit primary antibodies. The horseradish peroxidase enzyme conjugated to the antibody can be used for signal amplification in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl software, by GE Healthcare (2 mentions)
Anti myc tag polyclonal antibody pa1 981, by Thermo Fisher Scientific (2 mentions)
Nupage 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Alexa 488 conjugated goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Dapi dihydrochloride, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Merck Group (1 mentions)
4 protocols using goat horseradish peroxidase conjugated anti rabbit igg h l
1
Quantifying Myc-tagged Protein Levels
2
Quantifying Myc-tagged Protein Levels
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Protein extraction, separation by electrophoresis, transfer to membranes, membrane incubation with antibodies and washes, and chemiluminescence detection were carried out as described (22 (link)). An anti-Myc tag polyclonal antibody (PA1-981; Thermo Fisher Scientific) and goat horseradish peroxidase–conjugated anti-rabbit IgG (H + L) (Thermo Fisher Scientific) were diluted at 1:2,000 and 1:10,000 and used as primary and secondary antibodies, respectively. The intensities of the bands corresponding to the Myc-tagged fusion proteins were quantified using ImageQuant TL software (GE Healthcare, Chicago, IL). These intensities were then utilized to normalize the relative expression levels of the PhERF1-regulated metabolic genes, which were determined through RT-qPCR analysis.
3
Western Blot Analysis of Caspase-3 in Oligomycin A-Treated Hippocampal Neurons
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Oligomycin A treated 2 × 105 hippocampal neurons were collected in 1 × LDS buffer (50 mM Tris- HCl pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 1% beta-mercaptoethanol, 12.5 mM EDTA and 0.02% bromophenol blue).Proteins were separated using NuPAGE 12% Bis–Tris Protein Gels (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (PVDF, Bio- Rad Laboratories). After blocking in 5% w/v non-fat dry milk for 1 h at room temperature, membranes were probed with the following primary antibodies overnight at 4 °C26 : rabbit anti-Total Caspase 3 (Cell Signaling Technology, #9662, 1:3000), rabbit anti-Cleaved Caspase 3 (Cell Signaling Technology, #9664, 1:1000). This was followed by probing with corresponding secondary antibodies for 1 h at room temperature: horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) (Invitrogen, 656120, 1:6000). Images were collected using Bio-Rad Chemidoc Imaging System. Bands were quantified using Image J (National Institutes of Health, NIH, https://imagej.nih.gov/ij/links.html ).
4
Characterization and Differentiation of Murine Bone Marrow Mesenchymal Stem Cells
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The mBMSC culture and differentiation medium was purchased from Cyagen (China). The chondrocyte activator IL-1β was purchased from Sigma-Aldrich (USA). The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA). Flow cytometry anlysis was performed to identify the characterization of mBMSCs stained with FITC-conjugated or PE-conjugated anti-mouse CD44, CD45, CD90, and CD105 (BD, USA).
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