Total proteins were prepared by resuspending 1 × 106 cells in extraction buffer (50 mM Tris-HCl pH 7.6; 0.15 M NaCl; 5 mM EDTA; 16 Protease Inhibitors; 1% Triton X-100). One 40 s pulse of sonication (UP100H manual sonicator, Hielscher) at 40% amplitude was performed to allow dissociation of protein from chromatin and solubilization. Extracts were analyzed by SDS-PAGE using an 8% gel (37.5:1 Acryl/Bis Acrylamide). The following primary antibodies were used: Beta-Actin (Santa-Cruz sc1616, rabbit 1:4000), H3 total (Abcam ab1791, rabbit 1:6000), Lamin A/C (Santa Cruz sc-6215, goat 1:4000), Lamin B (Santa Cruz sc6216, goat 1:2000), progerin (13A4 mouse, Abcam 66587, mouse 1:1000), Ezh2 (AC22 Cell Signaling 3147S, mouse 1:1000), Bmi1 (D42B3 Cell signaling, rabbit 1:1000), H3K9me3 (Abcam ab8898, rabbit 1:1000), H3K27me3 (Millipore 07-449 rabbit 1:1000). HRP-conjugated secondary antibodies were revealed with the ECL chemiluminescence kit (ThermoFisher Scientific). The following secondary antibodies were used: Anti-Mouse IgG-Peroxidase (Sigma, A9044), Anti-Rabbit IgG-Peroxidase (Sigma, A9169), Anti-Goat IgG-Peroxidase (Sigma, A5420).
Anti goat igg peroxidase
Manufactured by Merck Group
Sourced in United States
Anti-Goat IgG-Peroxidase is a laboratory reagent used for detection and quantification of goat immunoglobulin G (IgG) in various immunoassays. It contains anti-goat IgG antibodies conjugated to the enzyme peroxidase, which catalyzes a colorimetric or luminescent reaction when exposed to a suitable substrate.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse igg peroxidase, by Merck Group (2 mentions)
Anti rabbit igg peroxidase, by Merck Group (2 mentions)
Ecl chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Total h3, by Abcam (1 mentions)
H3k27me3, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Lamin a c, by Santa Cruz Biotechnology (1 mentions)
H3k9me3, by Abcam (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Wet transfer system, by Bio-Rad (1 mentions)
Multiskan ascent 354, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
Anti flag m2 f1804, by Merck Group (1 mentions)
Imagequant las 4010 system, by GE Healthcare (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti goat igg peroxidase
1
Protein Extraction and Western Blot Analysis
2
IgG Cleavage Analysis by SDS-PAGE and Western Blot
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IgG cleavage was analysed by SDS‐PAGE followed by Coomassie blue staining or by western blot under denaturing or non‐denaturing conditions, as indicated. About 4–15% Mini‐PROTEAN® TGX™ Precast Protein Gels (#4561085, BioRad) were used for gel electrophoresis.
For Coomassie blue staining, a standard stain in Coomassie blue solution (0.1% Coomassie Brilliant Blue R‐250, 50% methanol and 10% glacial acetic acid) was performed.
For western blot analysis, protein was transferred using a wet transfer system (BioRad). After blocking with PBS/10% reconstituted skim milk powder, total IgG, scIgG, F(ab’)2 and Fab bands were detected with antibodies specific for each species (human, NHP: Captureselect™ biotin anti‐IgG‐CH1 conjugate, #7103202100, Thermo Fisher Scientific, Amsterdam, the Netherlands; mouse: goat anti‐mouse IgG Fab secondary Ab, #SA5‐10226, Thermo Fisher Scientific, Rockford, IL, USA), followed by either HRP conjugate (#SA10001, Thermo Fisher Scientific) or anti‐goat IgG‐peroxidase (#5420, Sigma‐Aldrich) as secondary Abs. Bands were revealed with SuperSignal West Pico Plus (#34580, Thermo Fisher Scientific) and analysed with an Odyssey imaging system Laia [(LI‐COR Biosciences (Lincon, NE, USA)].
For Coomassie blue staining, a standard stain in Coomassie blue solution (0.1% Coomassie Brilliant Blue R‐250, 50% methanol and 10% glacial acetic acid) was performed.
For western blot analysis, protein was transferred using a wet transfer system (BioRad). After blocking with PBS/10% reconstituted skim milk powder, total IgG, scIgG, F(ab’)2 and Fab bands were detected with antibodies specific for each species (human, NHP: Captureselect™ biotin anti‐IgG‐CH1 conjugate, #7103202100, Thermo Fisher Scientific, Amsterdam, the Netherlands; mouse: goat anti‐mouse IgG Fab secondary Ab, #SA5‐10226, Thermo Fisher Scientific, Rockford, IL, USA), followed by either HRP conjugate (#SA10001, Thermo Fisher Scientific) or anti‐goat IgG‐peroxidase (#5420, Sigma‐Aldrich) as secondary Abs. Bands were revealed with SuperSignal West Pico Plus (#34580, Thermo Fisher Scientific) and analysed with an Odyssey imaging system Laia [(LI‐COR Biosciences (Lincon, NE, USA)].
3
Optimized Immunoglobulin Quantification in Mucus
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Optimal test conditions were established based on the results obtained from two pools of positive and negative mucus samples. The final concentrations of somatic antigen used for the determination of specific immunoglobulin G (IgG) or IgA levels in mucus were 3.0 µg/ml or 5.0 µg/ml, respectively. Samples were diluted (1:100 for IgG or 1:25 for IgA) in PBS (0.8% w/v NaCl, 0.02% w/v KCl, 0.144% w/v Na2HPO4, 0.024% w/v KH2PO4; pH 7.2), and conjugate (anti-goat IgG-peroxidase [Sigma-Aldrich Inc., USA] or anti-goat IgA-peroxidase [Acris GmbH, Germany]) was diluted in PBS and used at a 1:1000 or 1:5000 dilution, respectively.
A citric acid-phosphate buffer containing 0.04% (w/v) o-phenylenediamine dihydrochloride (OPD) and 0.1% (v/v) H2O2 was used as substrate. All samples were analyzed in duplicate; the optical density (OD) was determined at a wavelength of 492 nm (Multiskan Ascent 354, Thermo Labsystems, USA) [22 (link)].
A citric acid-phosphate buffer containing 0.04% (w/v) o-phenylenediamine dihydrochloride (OPD) and 0.1% (v/v) H2O2 was used as substrate. All samples were analyzed in duplicate; the optical density (OD) was determined at a wavelength of 492 nm (Multiskan Ascent 354, Thermo Labsystems, USA) [22 (link)].
4
Western Blot Analysis of Protein Complexes
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The protein samples were added to SDS loading dye, heated to 95 °C, separated by SDS-PAGE and transferred to a nitrocellulose membrane (ThermoFisher Scientific). The antibodies anti-FLAG M2 (F1804; Sigma-Aldrich, 1:1000), anti-HA (Santa Cruz, sc-805, 1:200), anti-PKC (sc-216, Santa Cruz, 1:500), and anti-PAR6 (sc-33898, Santa Cruz, 1:500) were used as primary antibodies, anti-mouse IgG-Peroxidase (Sigma-Aldrich, 1:10,000), anti-rabbit IgG-Peroxidase (Sigma-Aldrich, 1:5000), and anti-goat IgG-Peroxidase (Sigma-Aldrich, 1:5000) for PAR3, aPKC, and PAR6, respectively, as secondary antibodies. The membranes were developed using SuperSignal West Pico or Femto Chemiluminescent Substrate (ThermoFisher Scientific). The detection was done by an ImageQuant LAS4010 system (GE Healthcare). The images were analyzed by ImageQuant TL toolbox version 8.1 following the company’s instructions and quantified by ImageStudio Lite (LI-COR, Lincoln, NE, U.S.A.).
5
Quantifying Mouse Brain Tumor Vascularity
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Resected mouse brains were sliced and fixed in 10% buffered formalin solution for 1 day and then embedded in paraffin. The presence of a tumor was determined by H&E staining. For mean vascular area (MVA) analysis, sections were incubated with goat anti‐rat CD34 polyclonal antibody (R&D Systems; #AF4117, 1:100) and then with the secondary antibody (anti‐goat IgG‐ peroxidase; Sigma‐Aldrich, A5420, 1:200) and detected with the 3,3ʹ‐diaminobenzidine substrate (Sigma‐Aldrich). Slides were counter‐stained with hematoxylin. Quantification of the CD34‐positive vascular area was performed using ImageJ software.31 MVA was calculated as the average of 5 vascular hot spots (500 × 500 μm2 in each spot) chosen in each sample.
6
Proteasome Inhibitors Regulate ORC1 Proteins
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To evaluate the effects of proteasome inhibitors on the regulation of ORC1 proteins, nuclei were extracted from 7 dps whole seedlings (ORC1b-GFP) or roots (ORC1a-GFP) treated as indicated, using Honda Buffer (0.44 M sucrose, 1.25% Ficoll, 2.5% Dextran T40, 20 mM Hepes HOK pH7.4, 10 mM MgCl2, 0.5% Triton X-100). 70 μg of nuclear proteins were loaded in a 6% Tris-glycine polyacrylamide gels to run SDS-PAGE and subsequent Western Blot. The proteins were transferred to a membrane, blocked 5% non-fat milk and then incubated with the primary antibody overnight at 4 °C (anti-GFP (Abcam ab5450) diluted 1:2000). After three washes the membrane was incubated with the secondary antibody for 1 h at room temperature (Anti-goat IgG -Peroxidase (Sigma A-5420) diluted 1:10000), washed again three times and proteins were detected using the kit Immobilon WB Chemiluminescent for HRP substrates (Millipore).
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