Whole cell extracts for SDS-PAGE were generated as follows (26 (link)): 1 × 106 BMDCs or B cells were washed with ice-cold PBS and resuspended in 50 μl lysis buffer mixed with Na3VO4, NaF, and PMSF. Afterwards, protein concentrations were determined by the BCA protein assay described above. Sample proteins were boiled in the presence of a loading dye mixed with SDS and 2-ME for 10 min at 95°C. Then, 20 μg of total protein per sample were separated on a 12.5% polyacrylamide gel and afterwards transferred onto nitrocellulose filters (Schleicher & Schüll/GE Healthcare), with a pore size of 0.2 μm with the wet blotting device “Mini-Protean II Cell and System” (BioRad). Membranes were incubated with primary antibodies against human CD83 (1G11, kindly provided by Elisabeth Kremmer, LMU Munich, Germany), murine CD83 (R&D Systems) or Beta-actin (AC-74; Sigma-Aldrich), followed by HRP-conjugated goat anti-rat IgG (Cell Signaling Technology), donkey anti-goat IgG (Promega), or goat anti-mouse IgG (Promega) antibodies. Detection was performed with the chemiluminescent ECL Prime Western Blotting Detection Reagent (Amersham, GE Healthcare) on a high performance chemiluminescence film (GE Healthcare).
Goat anti mouse igg
Manufactured by Promega
Sourced in United States
Goat anti-mouse IgG is a secondary antibody that recognizes and binds to mouse immunoglobulin G (IgG) molecules. It is commonly used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to detect and visualize the presence of mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Promega (2 mentions)
Enhanced chemiluminescence ecl detection kit, by Cytiva (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mini protean 2 cell and system, by Bio-Rad (1 mentions)
Beta actin ac 74, by Merck Group (1 mentions)
Hrp conjugated goat anti rat igg, by Cell Signaling Technology (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
High performance chemiluminescence film, by GE Healthcare (1 mentions)
Anti β tubulin, by Merck Group (1 mentions)
Supersignal chemiluminescent substrate, by Bio-Rad (1 mentions)
Aromatase, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Gapdh clone 0411, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit igg, by Cytiva (1 mentions)
Anti egfr, by Abcam (1 mentions)
Anti integrin α6, by Abcam (1 mentions)
Anti erk, by Abcam (1 mentions)
Anti pten, by Abcam (1 mentions)
Anti integrin β1, by Abcam (1 mentions)
Mouse anti human beta actin β actin antibody, by Cell Signaling Technology (1 mentions)
6 protocols using goat anti mouse igg
1
Western Blot Analysis of CD83 Expression
2
Western Blot Analysis of Trypanosome Proteins
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Cells (5 × 106) for sample preparation were harvested by centrifugation and washed in PBS (137 mM NaCl, 4 mM Na2HPO4, 1.7 mM KH2PO4, 2.7 mM KCl). Pellets were resuspended in urea cracking buffer (6 M urea, 10 mM Na2HPO4, 1% β-mercaptoethanol, pH 7) and boiled after the addition of loading sample buffer (67.5 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol, 5% β-mercaptoethanol). SDS-PAGE was performed, and polyvinylidene difluoride (PVDF) membranes were incubated with rabbit polyclonal anti-TbCDA (1:500) antibody generated against recombinant T. brucei CDA, rabbit polyclonal anti-TbdUTPase (1:75,000) antibody generated against recombinant T. brucei dUTPase, or mouse monoclonal anti-DCTD (1:1,000 [Santa Cruz Biotechnology]) or anti-β-tubulin (1:5,000 [Sigma]) antibodies. Bound antibodies were revealed by using goat anti-rabbit IgG (1:5,000) or goat anti-mouse IgG (1:3,000) antibodies (Promega) and the ECL enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).
3
Western Blot Analysis of Chikungunya Virus Proteins
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Cells were lysed with the addition of a radio-immunoprecipitation assay buffer in ice for 30 min. Cell lysates were centrifuged at 12,000 × g for 20 min at 4°C. The clarified supernatant protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, United States). The proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature using 5% bovine serum albumin in PBS-0.1%Tween 20, followed by overnight incubation at 4°C with the primary Ab against target proteins (anti-CD32; Santa Cruz Biotechnology, Dallas, TX, United States and in house antibodies against CHIKV E2). Blots were subsequently incubated with a 1:10,000 dilution of a horseradish peroxidase-conjugated secondary goat anti-rabbit IgG or goat anti-mouse IgG (Promega). The signals were detected using SuperSignal Chemiluminescent Substrate (Biorad, Herecules, CA, United States).
4
Quantitative Immunoblotting of Human Fibroblasts
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Whole-cell protein lysates (harvested in RIPA buffer) or cytoplasmic fractions from primary human dermal fibroblasts were resolved using 10% SDS PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Following blocking with 5% non-fat dry milk, membranes were incubated with the following primary antibodies: FN (clone EP5, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FN (clone F14, Abcam, Waltham, MA, USA), alpha tubulin (clone DM1A, Abcam, Waltham, MA, USA), Col IIIA1 (Proteintech, Rosemont, IL, USA), Col VA1 (clone C-5, Santa Cruz Biotechnology, Santa Cruz, CA, USA), aromatase (Invitrogen, Waltham, MA, USA), and GAPDH (clone 0411, Santa Cruz Biotechnology, Santa Cruz, CA, USA). This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG (Promega, Madison, WI, USA) and donkey anti-rabbit IgG (Cytiva, Malborough, MA, USA). After washing, immunoblots were developed with chemiluminescence reagents according to the manufacturer's protocol (Pierce, Rockford, IL, USA). Signal intensities were quantified by densitometry, normalized against GAPDH or alpha tubulin in each sample, and expressed as the magnitude of increase compared with controls. Densitometry was calculated using Image J v.1.53t [59] .
5
Ursolic acid and aspirin combination protocol
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Ursolic acid (UA, purity > 90%) was purchased from Xi'an Ocean Biological Engineering Co. (Xian, China). Aspirin (Asp, purity > 90%) was purchased from Aladdin Reagent Company (Shanghai, China). UA, Asp and Asp-UA were dissolved in dimethyl sulfoxide (DMSO) and were used in all experiments. PE-labeled mouse anti-human E-cadherin, vimentin, EpCAM and integrin α1, α3, α5, α6 and β1 integrin mAbs, PE/FITC mouse IgG1 kappa isotype control and APC mouse IgG1 kappa isotype control antibody were all purchased from Becton Dickinson (BD) PharmingenTM (New Jersey, USA). Mouse anti-human beta-actin (β-actin) antibody was purchased from Cell Signaling Technology (Danfoss, USA). Matrigel were obtained from BD BiocoatTM (New Jersey, USA). The primary anti-EGFR, anti-ERK, anti-MMP-2, anti-MMP-9, anti-COX-2, anti-E-cadherin, anti-β-catenin, anti-integrin α6, anti-integrin β1, anti-PTEN and anti-CD44 antibodies were obtained from Abcam Biotechnology (Cambridge, UK). The secondary antibody goat-anti-mouse-IgG or goat-anti-rabbit-IgG horseradish peroxidase was obtained from Promega (Madison, USA). All other reagents used in this study were of the highest purity commercially available.
6
Western Blot Analysis of TbHD82 Protein
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For western blot analysis, 5 x 106 cells were harvested, centrifuged and washed in 1 × PBS (137 mM NaCl, 4 mM Na2HPO4, 1.7 mM KH2PO4 and 2.7 mM KCl). Pellets were resuspended in urea buffer (6 M urea, 10 mM Na2HPO4, 1% β-mercaptoethanol, pH 7) and loading sample buffer (67.5 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol, 5% β-mercaptoethanol) in a 3:1 ratio, and boiled for 5 min at 99°C. Samples were subjected to electrophoresis in SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes, which were incubated with a rabbit polyclonal anti-TbHD82 (1:10,000) antibody generated against recombinant TbHD82 (Yague-Capilla et al., 2021 (link)). The mouse monoclonal anti-Tbβ-tubulin (1:10,000 [Sigma]) antibody was used as loading marker. Goat anti-rabbit IgG (1:5,000) or goat anti-mouse IgG (1:3,000) HPRT-conjugated antibodies (Promega) were used as secondary antibodies and films were developed using the ECL enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).
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