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Anti rabbit secondary antibody conjugated with horseradish peroxidase

Manufactured by Cell Signaling Technology
Sourced in United States

The Anti-rabbit secondary antibody conjugated with horseradish peroxidase is a laboratory reagent used to detect and visualize target proteins in various immunoassays. It binds to primary antibodies that are raised against rabbit antigens and the conjugated horseradish peroxidase enzyme catalyzes a colorimetric reaction, enabling the detection of the bound target proteins.

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4 protocols using anti rabbit secondary antibody conjugated with horseradish peroxidase

1

Western Blot Analysis of HA-tagged Proteins

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The protein samples were separated by SDS-PAGE using 10 % acrylamide gel and transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with 1 × TBS (Tris-buffered saline with 0.1 % (v/v) Tween 20) containing 6% (w/v) skim milk for 30 min. Then, the membrane was incubated with an anti-HA antibody (1:1000 dilution) at 4 °C overnight. After washing with 1 × TBS-T three times, the membrane was incubated with an anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:5000 dilution) (Cell signaling Technology, cat no: 7074S) at 4 °C for 4 h. After washing with 1 × TBS-T three times, the membrane was immersed in ECL reagents (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the chemiluminescence images were captured using the ChemiDocTM XRS+ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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2

Quantitative Western Blot Analysis

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Total protein was extracted from cells using RIPA buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (BioDee, Beijing, China) (100:1) and phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) (100:1). Proteins were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (GenScript, Nanjing, China). The proteins were transferred to polyvinylidene difluoride membranes, and then blocked with 5% non-fat milk for 1 h. The membranes were incubated with the primary antibodies CPT2 (1:1,000, NBP1-32226; Novus, Columbia, USA) and beta-actin (1:5,000, NB600-532SS; Novus, USA) for 1 h, and washed with PBST (Solarbio, China) three times, for 5 min each time. The membranes were then incubated with anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:1,500, 7074; Cell Signaling Technology, Boston, MA, USA) for 1 h at room temperature, and washed three times using PBST. Protein bands were visualized using an enhanced chemiluminescence system (GE Healthcare, Fairfield, CT, USA).
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3

Calcium Signaling Pathway Analysis

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Acetomethylated Fluo-4, Calcium Orange, and BAPTA were purchased from Invitrogen (Carlsbad, CA). Forskolin was obtained from Sigma-Aldrich (St. Louis, MO). d-Luciferin and pGL4.10 [luc2] vector were from Promega (Madison, WI). Thapsigargin, ATP, 11-cis-retinal, pertussis toxin, and other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Antibodies against ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), JNK/SAPK, phospho-JNK/SAPK (Thr183/Tyr185), and anti-rabbit secondary antibody conjugated with horseradish peroxidase were obtained from Cell Signaling Technology (Beverly, MA). Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA), and the ECL Prime Western Blotting Detection System was from GE Healthcare UK Ltd (Buckinghamshire, England).
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4

Quantifying NF-κB Activation via Western Blot

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For the western blotting of NF-κB p65 and phospho-p65, 20 μg of whole cell lysates was used for SDS polyacrylamide gel-running, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using Pierce Power Blotter (Thermo Fisher Scientific) at 110 V (80 mA) for 80 min. The membrane was incubated in 5% skim milk/0.1% TBS-Tween 20 at room temperature for 1 h, followed by incubation with a 1:1,000 final diluted NF-κB p65 (#8242, Cell Signaling Technology) and phospho-p65 antibody (Ser536, #3033, Cell Signaling Technology) and then an anti-rabbit secondary antibody conjugated with horseradish peroxidase (#7074, Cell Signaling Technology). The control for loading was assessed using a β-actin antibody (at a dilution of 1:1,000, SC-47778, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States).
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