A total of 1.5 μg urine proteins were separated on 12 % SDS-PAGE and blotted onto nitrocellulose membranes, that were first blocked with 5 % non-fat milk and subsequently incubated overnight at 4 °C with the following primary antibodies (all from Abcam, Cambridge, UK): anti-Prostaglandin D Synthase (rabbit polyclonal, 1:500); anti-Uromucoid (rabbit polyclonal, 1:500); anti-Alpha-1-microglobulin (rabbit monoclonal, 1:1000); anti-Cystatin C (rabbit monoclonal, 1:500). Membranes were then incubated with a solution containing 1:2000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (DakoCytomation, Denmark). Target bands were visualized using a mix of peroxidase solution plus a luminol enhancer solution (WesternSure™ PREMIUM Chemiluminescent substrate). Results acquisition and band densitometric analysis (represented by arbitrary units, AU), were performed using the C-DiGit® Blot Scanner (LI-COR Biosciences, NE, USA) and the QuantityOne image analysis software (Bio-Rad). Human serum sample was used as positive (or negative) control.
Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody
Manufactured by Agilent Technologies
Sourced in Denmark
The Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody is a laboratory reagent that is used to detect and quantify the presence of rabbit primary antibodies in various immunoassays. The HRP enzyme attached to the secondary antibody facilitates the detection of the target rabbit antibodies through colorimetric or chemiluminescent signals.
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2 protocols using horseradish peroxidase hrp conjugated goat anti rabbit secondary antibody
1
Urine Protein Immunoblotting Analysis
2
Western Blot Analysis of TRPC4 Protein
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Protein samples were prepared from intact thoracic aorta by homogenization in lysis solution (Camiolo buffer, 75 mM potassium acetate, 300 mM NaCl, 10 mM EDTA, 100 mM L-arginine basic salt, and 0.25% Triton-X 100, protease inhibitor mix). Protein concentrations were determined using Bradford assay. Proteins were separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to membranes (Immobilon-P polyvinylidene difluoride, Millipore) at 25 °C for 2 h at 15 V. Following 2-h blocking (with 5% skimmed milk in Tris-buffered saline with Tween-20), membranes were incubated with TRPC4 primary antibody (1:200, Alomone Laboratories) and anti-β-actin (1:1,500, Abcam Ltd.) overnight at 4 °C, then with horseradish peroxidase (HRP)-conjugated goat antirabbit secondary antibody (DakoCytomation; 1:1,500) for 1 h at room temperature. ECL Plus Detection kit (Amersham Biosciences) was used to visualize bands, and the optical density of each blot was normalized to that of β-actin analyzed within the same lane and represented as relative optical density.
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