Horseradish peroxidase conjugated goat anti rabbit igg

Manufactured by Bioss Antibodies

Sourced in China

Horseradish peroxidase-conjugated goat anti-rabbit IgG is a secondary antibody conjugated with the enzyme horseradish peroxidase. It is designed to bind to and detect rabbit primary antibodies in various immunoassay applications.

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Lab products found in correlation

Immun star westernc kit, by Bio-Rad (1 mentions) Profinity imac ni charged resin, by Bio-Rad (1 mentions) Bicinchoninic acid protein kit, by Thermo Fisher Scientific (1 mentions) Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions) Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions) Ecl reagent, by Beyotime (1 mentions) Ecl western blot detection kit, by Beyotime (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Las 1000, by Fujifilm (1 mentions)

5 protocols using horseradish peroxidase conjugated goat anti rabbit igg

Purification and Antibody Production of HtrA and PotD

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The htrA gene was amplified from genome of H. parasuis SC1401 with primers P19 and P20 and cloned into the NcoI and XhoI sites of pET22b to form plasmid pET22b-htrA, which was expressed in E. coli BL21 (DE3). The recombinant HtrA was purified by metal affinity chromatography using Profinity IMAC Ni-Charged Resin (biorad) according to the manufacturer’s protocol. The generation and purification of recombinant PotD were also performed as described above. The production of rabbit antisera against recombinant HtrA and PotD was performed as described [24 (link)].
For western blotting analysis, the whole-cell extract of parental strain and mutant strains was analyzed by 12% SDS-PAGE and electrotransferred to a nitrocellulose membrane. After being blocked with 5% nonfat milk in PBST (phosphate-buffered saline containing 0.05% Tween 20) at room temperature (RT) for 30 min, the membrane was incubated at RT for 1h with rabbit antiserum against recombinant HtrA or PotD produced on the above as the primary antibody. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Bioss, China) were used as the secondary antibody. The membrane was developed with Immun-Star Western C Kit (biorad, USA) according to the manufacturer’s instructions.

Zhang L., Li Y., Dai K., Wen X., Wu R., Huang X., Jin J., Xu K., Yan Q., Huang Y., Ma X., Wen Y, & Cao S. (2015). Establishment of a Successive Markerless Mutation System in Haemophilus parasuis through Natural Transformation. PLoS ONE, 10(5), e0127393.

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Quantitative Western Blot Analysis

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Western blot assay was performed to measure levels of proteins of interest. Specimens were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane by a transfer apparatus at 300 mA for 2.5 hours. The membrane was blocked with 5% non-fat milk before incubation in 1% bovine serum albumin solution containing the following primary rabbit-anti-rat polyclonal antibodies overnight at 4°C: anti-phosphatase and tensin homolog deleted on chromosome ten (PTEN) (1:500), anti-Akt (1:500), anti-p-Akt (1:500), anti-Bcl-2 (1:500), anti-Bax (1:500), anti-cleaved caspase-3 (1:500) and anti-β-actin (1:1000). All primary antibodies were obtained from Wanleibio (Shenyang, China). After washes with Tris-buffered saline containing Tween, membranes were cultured with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Bioss, Woburn, MA, USA) for 45 minutes at 37°C and detected with an enhanced chemiluminescence system. ImageJ software (NIH, Bethesda, MD, USA) was used to analyze optical densities of immunoreactive bands. Protein expression was normalized to that of the loading control (β-actin).

Lu X., Zhang H.Y, & He Z.Y. (2020). MicroRNA-181c provides neuroprotection in an intracerebral hemorrhage model. Neural Regeneration Research, 15(7), 1274-1282.

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PC12 Cell Lysis and Western Blot

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PC12 cells were lysed in radio immunoprecipita-tion assay buffer (Beyotime, Beijing, China) containing 1% phenylmethylsulfonyl fluoride (Solarbio, Guangzhou, China) for 30 minutes. Lysates were centrifuged at 12,000 × g at 4°C for 15 minutes. Protein concentrations were determined using a bicinchoninic acid protein kit (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were separated via 5–15% SDS-PAGE electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, which were incubated in 1% bovine serum albumin solution with the following primary rabbit-anti-rat polyclonal antibodies overnight at 4°C: anti-PTEN (1:500), anti-Akt (1:500), anti-p-Akt (1:500), anti-Bcl-2 (1:500), anti-Bax (1:500), an-ti-cleaved caspase-3 (1:500), and anti-β-actin (1:1000). After washes with Tris-buffered saline containing Tween, membranes were cultured with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Bioss) for 45 minutes at 37°C.

Lu X., Zhang H.Y, & He Z.Y. (2020). MicroRNA-181c provides neuroprotection in an intracerebral hemorrhage model. Neural Regeneration Research, 15(7), 1274-1282.

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Western Blot Analysis of Lipid Metabolism Proteins

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Following the incubation with the different treatments, SDS buffer was used to extract total proteins from the harvested cells which were washed twice and collected in ice-cold PBS. The untreated cells were used as control. Equal amounts of total proteins (100 μg/lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (6%) and transferred to a Polyvinylidene Fluoride (PVDF) membrane. After blocking with a mixture of 5% skimmed milk/Tris-buffered saline Tween 20 (TBST), the membranes were incubated overnight at 4˚C with the primary antibody rabbit against sterol regulatory element-binding proteins-1 (SREBP1) (bs-1402R), carnitine palmitoyltransferase (CPT1A) (bs-2047R), microsomal triglyceride transfer protein (MTP) (bs-5083R) antibodies (1:1,000; Beijing Biosynthesis Biotechnology, China); antibody information was listed in Supplement materials S-Table2. Following three consecutive washes in TBST (0.05%), the membranes were incubated with the goat anti-rabbit horseradish peroxidase-conjugated IgG at 1:2,000 (Beijing Biosynthesis Biotechnology) for another 2 h at room temperature. The results were normalized to α-Tubulin (bs-0519R) (Beijing Biosynthesis Biotechnology) protein levels. Protein expression levels were finally visualized using enhanced chemiluminescence (ECL) reagents (Beyotime Institute of Biotechnology, China).

Wei R., Deng D., Teng Y., Lu C., Luo Z., Abdulai M., Liu H., Xu H., Li L., Hu S., Hu J., Wei S., Zeng X, & Han C. (2022). Study on the effect of different types of sugar on lipid deposition in goose fatty liver. Poultry Science, 101(4), 101729.

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Western Blot Analysis of ACCα in Hepatocytes

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Hepatocytes were washed twice and collected in ice-cold PBS. Total protein extracts were obtained using a reducing SDS buffer containing 50 mmol/L Tris-HCl (pH 6.8), 100 mmol/L DTT, 2% SDS, and 10% glycerol. Protein concentrations were determined for diluted samples using the Bradford procedure. Equal amounts of protein (100 μg) were separated by 6% SDS-PAGE and transferred onto membranes. The membranes were blocked in a TBS solution containing 5% nonfat dry milk and incubated with an antibody against ACCα (1:1000 dilution; Beijing Biosynthesis Biotechnology, China). A goat anti-rabbit horseradish peroxidase-conjugated IgG (1:2000; Beijing Biosynthesis Biotechnology, China) was used as the secondary antibody, and the signals were detected using an ECL western blot detection kit (Beyotime Institute of Biotechnology, China). After analysis, the membranes were blotted with ananti-α-tubulin antibody (1:1000; Beijing Biosynthesis Biotechnology, China) to normalize for protein content. The blot images were digitized using a luminescent image analyzer (LAS-1000, Fuji Photo Film). The protein bands were quantified using Quantity One software (Bio-Rad). The relative ACCα protein level was determined based on the value of ACCα expression divided by the value of α-tubulin expression. Each individual experiment was repeated three times and averaged.

Han C., Wei S., He F., Liu D., Wan H., Liu H., Li L., Xu H., Du X, & Xu F. (2015). The Regulation of Lipid Deposition by Insulin in Goose Liver Cells Is Mediated by the PI3K-AKT-mTOR Signaling Pathway. PLoS ONE, 10(5), e0098759.

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