Protein concentrations were determined by using a BCA kit (iNtRon Biotechnology). Total proteins (20 µg per sample) were loaded to each lane of 12% SDS-PAGE, electrophoresed, and transferred to PVDF membranes (Bio-Rad Laboratories, USA). Following transfer, membranes were blocked with TBST [100 mM Tris-HCl (pH 7.6), 0.8% NaCl, and 0.1% Tween-20] containing 5% skim milk (BD Biosciences, USA). The blocked membranes were incubated with diluted rabbit anti–ZO-1 (1:2,000; Thermo Scientific, USA), mouse anti-occludin (1:2,000; Thermo Scientific), and rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, USA) primary antibodies at 4℃ overnight. After further washing, membranes were incubated with peroxidase-labeled anti-rabbit and anti-mouse secondary antibodies (Vector, USA). Immunoreactive signals were detected by using an enhanced chemiluminescence reagent (Abclon, Korea) and recorded by using a MicroChemi 4.2 system (DNR Bio-Imaging Systems, Israel).
Peroxidase labeled anti rabbit and anti mouse secondary antibodies
Manufactured by Vector Laboratories
Sourced in United States
Peroxidase-labeled anti-rabbit and anti-mouse secondary antibodies are laboratory reagents designed for use in various immunodetection techniques. These antibodies are capable of binding to primary antibodies raised in rabbit or mouse, respectively, and are conjugated with the enzyme peroxidase. The peroxidase label allows for the visualization and amplification of the target antigen signal in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti zo 1, by Thermo Fisher Scientific (1 mentions)
Mouse anti occludin, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Skim milk, by BD (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Bca kit, by iNtRON Biotechnology (1 mentions)
Microchemi 4.2 system, by DNR Bio-Imaging Systems (1 mentions)
Gfp trap magnetic beads, by Proteintech (1 mentions)
Anti gfp, by Roche (1 mentions)
2 protocols using peroxidase labeled anti rabbit and anti mouse secondary antibodies
1
Western Blot Analysis of Tight Junction Proteins
2
Co-immunoprecipitation of HPL-1 and HPL-2
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For co-immunoprecipitation assays, frozen eggs from transgenic lines carrying hpl-1::GFP and hpl-2::GFP (prepared by hypochlorite treatment) were reduced into powder using a mortar and pestle. The powder was resuspended in IP buffer B (described in GFP pull-down section) and 2 mg of total proteins were incubated with blocked magnetic particles beads (bmp, Chromotek) overnight at 4°C. Pre-cleared soluble fractions were collected and incubated with Magnetic GFP-Trap beads (Chromotek) for 2 h at 4°C. Beads were washed five times in IP buffer B, boiled in SDS-sample buffer and analyzed by SDS-PAGE followed by western blotting. Antibodies used in this experiment were: anti-SPR-5 (sc-68340, Santa Cruz Biotechnology) at 1:100, anti-GFP (Roche, 11814460001) at 1:5000 and peroxidase-labeled anti-rabbit and anti-mouse secondary antibodies (Vector) at 1:10 000.
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