For Western blot analysis, whole-cell lysate and CM samples were subjected to SDS-PAGE and subsequently transferred to Immobilon PVDF membranes (Millipore). Membranes were blocked in 5% milk for 1 h prior to overnight primary antibody incubation. The primary antibodies used were anit-NID1 (1:1000; Abcam, ab133686), anti-FAK and anti-phospho-FAK (Tyr397) (1:1000; Cell Signaling Technology FAK antibody sampler kit, 9330), anti-integrin β1 antibody (EP1041Y) (1:1000; Abcam, ab52971), anti-integrin αV antibody (EPR16800) (1:5000; Abcam, ab179475), anti-integrin β3 antibody (EPR2342) (1:1000; Abcam, ab119992), and anti-integrin α3 antibody (1:1000; Abcam, ab190731). Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:2000 dilution; GE Healthcare) or anti-rabbit secondary antibody (1:2000 dilution; GE Healthcare) for 1 h, and chemiluminescence signals were detected by ECL substrate (GE Healthcare). Coomassie blue staining of the membrane was used to assess equal loading of CM samples. Gel quantifications were performed using ImageJ (National Institutes of Health).
Horseradish peroxidase hrp conjugated anti mouse secondary antibody
Manufactured by GE Healthcare
Sourced in United Kingdom, Brazil
Horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody is a laboratory reagent used to detect and visualize target proteins in various immunoassays, such as Western blotting and ELISA. The antibody binds to mouse primary antibodies and the HRP enzyme catalyzes a colorimetric or chemiluminescent reaction, allowing for the identification and quantification of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Ecl substrate, by GE Healthcare (2 mentions)
Anti rabbit secondary antibody, by GE Healthcare (2 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Anti γh2ax, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Ecl western blotting system, by GE Healthcare (1 mentions)
Pure nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti psmad3, by Thermo Fisher Scientific (1 mentions)
Anti zeb1, by Fortis Life Sciences (1 mentions)
Anti cdh1, by BD (1 mentions)
Anti p smad2, by Cell Signaling Technology (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti vimentin, by BD (1 mentions)
4 protocols using horseradish peroxidase hrp conjugated anti mouse secondary antibody
1
Western Blot Analysis of Integrin Proteins
2
Quantification of ATM and γH2AX in Fetal Liver Hematopoietic Stem Cells
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Aliquots of 1 × 106 FL-HSCs isolated from five fetal livers
from pregnant mice on day 13.5 were washed with PBS and lysed in RIPA buffer
(150 mM NaCl, 1.0% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 5 mM EDTA, and 10
mM Tris-HCl [pH 7.4]) containing protease inhibitors. Protein concentration was
measured using the DC Protein Assay Kit (Bio-Rad, Richmond, CA, USA). After
boiling with sample buffer, 30 μg of protein was subjected to SDS-PAGE
and transferred to a membrane. Blots were probed with anti-ATM (4D2),
anti–phospho-ATM Serine 1981 (Cell Signaling Technology, Danvers, MA,
USA), anti-γH2AX (Cell Signaling Technology), or β-actin
(Sigma-Aldrich) antibody. Primary antibodies were detected using horseradish
peroxidase (HRP)-conjugated anti-mouse secondary antibody (GE Healthcare, Little
Chalfont, UK).
from pregnant mice on day 13.5 were washed with PBS and lysed in RIPA buffer
(150 mM NaCl, 1.0% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 5 mM EDTA, and 10
mM Tris-HCl [pH 7.4]) containing protease inhibitors. Protein concentration was
measured using the DC Protein Assay Kit (Bio-Rad, Richmond, CA, USA). After
boiling with sample buffer, 30 μg of protein was subjected to SDS-PAGE
and transferred to a membrane. Blots were probed with anti-ATM (4D2),
anti–phospho-ATM Serine 1981 (Cell Signaling Technology, Danvers, MA,
USA), anti-γH2AX (Cell Signaling Technology), or β-actin
(Sigma-Aldrich) antibody. Primary antibodies were detected using horseradish
peroxidase (HRP)-conjugated anti-mouse secondary antibody (GE Healthcare, Little
Chalfont, UK).
3
Western Blot Analysis of Hippocampal EAAT1 and EAAT2
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Hippocampal slices for electrophoresis/Western blot analysis were directly homogenized in electrophoresis sample buffer at pH 6.8 (containing 62.5 mM Tris-HCl, 2% (w/v) SDS, 5% (w/v) β-mercaptoethanol, 10% (v/v) glycerol, 0.002% (w/v) bromophenol blue) and boiled for 2 min. Protein samples (20μg per lane) were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using a semidry blotting apparatus (1.2mA/cm2, 1 h; [36 (link)]. The membranes were blocked overnight with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS; 10mM Tris, 150mM NaCl, pH 7.5) and then incubated for 3 h with an anti-EAAT 1 and anti-EAAT 2 (antibody diluted 1:1000 and 1:200, respectively, in TBS containing Tween-20 and 2% BSA). Next, membranes were incubated for 2h at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (GE Healthcare, Sao Paulo—Brazil). The chemiluminescent reactions were developed using luminol as the substrate (ECL Western Blotting System, GE Healthcare, Sao Paulo—Brazil) and registered on radiographic film. The immune content of EAAT 1 and EAAT 2 was determined as a ratio of the optical density (OD) of the β-actin protein band [(EAAT1/2)/OD of the β-actin band]. The bands were quantified using Scion Image software.
4
Western Blot Analysis of EMT Markers
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SDS lysis buffer (0.05 mM Tris-HCl, 50 mM BME, 2% SDS, 0.1% Bromophenol blue, 10% glycerol) was used to collect protein from cells. Samples were heat denatured. Protein was equally loaded, separated on a 10% SDS-page gel, transferred onto a pure nitrocellulose membrane (BioRad), and blocked with 5% milk. Primary antibodies for immunoblotting included anti-pSMAD2 (1:1000, Cell Signaling, 3108), anti-pSMAD3 (1:250, ThermoFisher, 44-246 G), anti-SMAD2/3 (1:1000, Cell Signaling, 3102), anti-CDH1 (1:5000, BD Biosciences, 610181), anti-CDH2 (1:2000, BD Biosciences, 610920), anti-ZEB1 (1:3000, Bethyl, A301-922A), anti-Vimentin (1:5000, BD Biosciences, 550513), and anti-β-Actin (1:5000, Abcam, ab8227) for loading control. Membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:5000, GE Healthcare) or anti-rabbit secondary antibody (1:5000, GE Healthcare) for 1 h, after which chemiluminescent signals were detected using ECL substrate (GE Healthcare).
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