Samples of His‐tagged hCLDN‐4 with or without sodium dodecyl sulfate and 2‐mercaptethanol (each containing 50 μg protein in 2 μL of phosphate‐buffered saline) were spotted onto polyvinylidene fluoride membrane (GE Healthcare). After blocking of the membranes with Tris‐buffered saline containing 5% skim milk and 0.05% Tween‐20, the membranes were treated with 1 μg/mL of 5D12 or 100‐fold‐diluted mouse anti‐His tag antibody (Life Technologies) as a primary antibody at 4°C for 12 h and then with 1000‐fold‐diluted horseradish‐peroxidase–conjugated anti‐rat IgG (Jackson ImmunoResearch, West Grove, PA) or horseradish‐peroxidase–conjugated anti‐mouse IgG (Jackson ImmunoResearch), respectively, as a secondary antibody at room temperature for 1 h. After the membrane had been washed with Tris‐buffered saline containing 0.05% Tween‐20, the antibody‐reacted dots were detected by using a chemiluminescence imaging system (ECL Western Blotting Detection Reagent and LAS‐4010 ImageQuant, GE Healthcare).
Horseradish peroxidase conjugated anti rat igg
Manufactured by Jackson ImmunoResearch
Horseradish-peroxidase–conjugated anti-rat IgG is a secondary antibody reagent used to detect the presence of rat immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with the enzyme horseradish peroxidase, which can be used as a reporter molecule to generate a colorimetric or chemiluminescent signal upon binding to the target rat IgG.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (1 mentions)
Mouse anti his tag antibody, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Stain free technology, by Bio-Rad (1 mentions)
Ecl select, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
3 protocols using horseradish peroxidase conjugated anti rat igg
1
Quantification of His-Tagged hCLDN-4 Proteins
2
Western Blot Analysis of Perforin Expression
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Cells were collected and lysed in M2 lysis buffer (20 mM Tris, pH7.0, 0.5% NP40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 05 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 μg/mL Leupeptin), then resolved by SDS-PAGE. Blots were probed with anti-Perforin (CB5.4, Abcam; RRID:AB_302236 ) and anti-actin (AC40, Sigma; RRID:AB_476730 ), and visualized using enhanced chemiluminescence (ThermoScientific) with horse-radish peroxidase conjugated anti-rat IgG (712-035-150, Jackson ImmunoResearch; RRID:AB_2340638 ) or anti-mouse IgG (401215, Calbiochem; RRID_AB:2686924).
3
Protein Extraction and Immunoblotting Analysis
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Cells were lysed in lysis buffer [137 mM NaCl, 10% glycerol, 20 mM Tris–HCl pH 8.0, 2 mM ethylenediaminetetraacetic acid pH 8.0, 1% Igepal, 5 μl protease inhibitor cocktail (Sigma) for 20 min on ice. After centrifugation (15 min at 17 000 g, 4°C) the protein content in the soluble fraction was determined according to the Bradford method. A total of 10–20 μg protein were loaded onto precast gels with stain free technology (Bio-Rad). Total lane protein was visualized as loading control with the ChemiDoc Imaging System (Bio-Rad) prior transfer onto PVDF membranes (Bio-Rad). The primary antibody Roquin-1/-2 (Millipore 3F12) and the secondary antibody Horseradish peroxidase-conjugated anti-rat IgG (Jackson Immunoresearch) were used. Blots were developed with the ECL select (Life technologies). Imaging was performed with the ChemiDoc Imaging System and quantified using Image Lab Software (Bio-Rad).
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