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Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody

Manufactured by Cell Signaling Technology
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Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody is a laboratory reagent used to detect the presence of rabbit primary antibodies in various immunoassays. The secondary antibody is conjugated with the enzyme horseradish peroxidase, which can catalyze a colorimetric or chemiluminescent reaction for signal detection.

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10 protocols using horseradish peroxidase conjugated goat anti rabbit igg secondary antibody

1

Protein Quantification and Western Blot Analysis

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Total protein was isolated from cells using radioimmunoprecipitation assay buffer and quantified using the bicinchoninic acid method. Equal amounts of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto a polyvinylidene fluoride (PVDF) membrane. Subsequently, the PVDF membrane was blocked with 5% fat-free milk for 1 h at 25°C and incubated with specific primary antibodies against STAT3 (1:1000; SAB4502078; Sigma-Aldrich), matrix metalloproteinase (MMP)-2 (1:1000; 40,994; Cell Signaling Technology), MMP-9 (1:1000; 13,667; Cell Signaling Technology), and β-actin (1:1000; 3700; Cell Signaling Technology) at 4°C overnight. After washing three times with TBST for 10 min each, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000; 14708s; Cell Signaling Technology) for 1 h at 25°C. The protein bands were visualized using enhanced chemiluminescence reagent and the gray value was determined using an imaging system (Bio-Rad).
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2

Immunoblotting Protocol for Protein Detection

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Immunoblotting was carried out as described previously [76 (link)]. The following antibodies were used: mouse anti-Flag M2 monoclonal antibody (1:1500 dilution; Sigma #F3165), rabbit anti-ATF4 polyclonal antibody (1:5000 dilution; Santa Cruz Biotechnology sc-200, Santa Cruz, CA, USA) and rabbit anti-β-Tubulin polyclonal antibody (1:2500 dilution; Abcam #ab6046, Cambridge, UK). Depending on the species of the primary antibody, the secondary antibody used was either horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:10,000 dilution; Thermo Scientific #31430) or horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000 dilution; Cell Signaling Technology #7074, Danvers, MA, USA). Blots were treated with Immobilon chemiluminescent reagent (EMD Millipore, Burlington, MA, USA) and proteins were visualized either on autoradiography film or with ChemiDoc XRS+ detection system (BioRad). Uncropped western blot images are presented in Figures S2 and S3.
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3

Western Blot Analysis of Cerebral CBS

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Proteins were extracted from brain tissue, briefly, brain tissue was triturated in a tissue mortar with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) at 4°C for 10 min, the brain lysate solution was centrifuged at 15,000 × g and 4°C for 10 min. Following centrifugation, the supernatant was collected. Protein (80 µg) was loaded onto each lane, separated by 10% SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat milk powder for 30 min and subsequently probed with rabbit polyclonal CBS (1:1,000; cat. no. H00000875-D01P; Abnova, Taipei, Taiwan) and rabbit monoclonal GAPDH (1:1,000; cat. no. 2118S; Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies for 2 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20,000; cat. no. E030120; EarthOx Life Sciences, Millbrae, CA, USA) for 1 h at room temperature. Membranes were developed with an Enhanced Chemiluminescence reagent (LumiGold; SignaGen Laboratories, Rockville, MD, USA) and a Tanon 5200 Gel Imaging System.
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4

M1/M2 Macrophage Protein Expression Analysis

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M1/M2 polarization‐related protein expression was analyzed using Western blot. Briefly, macrophages were isolated from the peritoneal cavity of rats as previously described.17 The isolated macrophages were homogenized as previously described.18 The protein concentration was determined by a Bradford assay (Bio‐Rad Laboratories, Hercules, CA, USA). Ten microgram aliquots of cell homogenate were loaded per well on a 10% sodiumdodecyl sulfate polyacrylamide gel electropheresis (SDS‐PAGE) gel. After transferring the protein to the membranes, they were then blocked with 5% non‐fat dried milk for 1 h and incubated with anti‐CD38 (1:1000 dilution), Egr2 (1:1000 dilution), IRF4 (1:1000 dilution), IRF5 (1:1000 dilution) and β‐actin antibody (1:1500 dilution) (Cell Signaling, Danvers, MA, USA) overnight at 4 °C followed by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (Cell Signaling) for 2 h at room temperature. An electro‐chemi‐luminescence (ECL) chemiluminescence substrate (Amersham, Little Chalfont, UK) was used to visualize the immune reaction. The images were taken using ChemiDoc XRS+ system (Bio‐Rad Laboratories, Hercules, CA, USA) and analyzed using Image Lab 4.1 (Bio‐Rad Laboratories, Hercules, CA, USA).
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5

Quantifying NECTIN-1 protein levels

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Cellular proteins were extracted using cold RIPA lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dried milk and then incubated with primary antibodies (anti–NECTIN-1, 1:1000; Abcam) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated goat anti-rabbit IgG secondary antibody (1:1000; Cell Signaling Technology, Danvers, USA). Chemiluminescence solution (MilliporeSigma, Burlington, USA) was used to reveal the bands. Representative blots from at least three independent experiments were shown.
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6

Chondrocyte Nuclear p-p65 Imaging

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After treating with 10% FBS, Second-generation chondrocytes cultured with DMEM supplemented with 0.05% FBS were treated with 10 ng/ml IL-1β, or co-incubated with 10 ng/ml IL-1β and 1 mM AG for 2 h at 37˚C. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at 37˚C. The cells were washed with PBS, blocked with 10% goat serum (cat. no. AR1009; 0.3% Triton, 1:10; Boster Biological Technology) for 1 h at room temperature and rinsed with PBS. Subsequently, the cells were incubated with primary antibodies against p-p65 (cat. no. 3033; 1:100; Cell Signaling Technology, Inc.) overnight at 4˚C. Following primary incubation, the cells were gently washed and then incubated with a horseradish peroxidase-conjugated secondary antibody goat anti-rabbit IgG (cat. no. ZB-2301; 1:250; OriGene Technologies, Inc.) for 2 h at 37˚C. Subsequently, the cells were stained with DAPI (Beyotime Institute of Biotechnology) at room temperature for 15 min. Cells were rinsed and observed under a fluorescence microscope in six randomly-selected fields (magnification, x400).
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7

Western Blot Analysis of Myc Protein

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Total protein was obtained using RIPA buffer with cocktail inhibitors (Cell Signaling Tech.). Protein concentration was measured using a BCA kit (Pierce, USA). Equal amounts of protein were separated on a15% gel and then transferred to 0.22μm PVDF membranes (AmerSham, USA). The membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 buffer (TBST) for 2 h and then incubated overnight at 4°C with the following primary antibodies: anti-Myc (1:1000, Cell Signaling Tech.) and anti-β-actin (1:1000, Cell Signaling Tech.). Then, the membranes were washed 3 times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, 1:5000 or goat anti-mouse IgG, 1:5000,Cell Signaling Tech.) for 1 hatroom temperature. Blots were developed using a chemiluminescence kit (Pierce) and exposed to X-ray film. The bands on the film were scanned and analyzed with Quality One software (Bio-Rad).
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8

Western Blot Analysis of FAK Protein

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Total protein was obtained using RIPA buffer with cocktail inhibitors (Cell Signaling Tech, USA). Protein concentration was measured using a BCA kit (Pierce, USA). Equal amounts of protein were separated on a 15% gel and then transferred to 0.22 μm PVDF membranes (AmerSham, USA). The membranes were blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 buffer (TBST) for 2 h and then incubated overnight at 4 °C with the following primary antibodies: FAK-antibody (1:1000, Cell Signaling Tech, USA) and anti-β-actin (1:1000, Cell Signaling Tech, USA). Then, the membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, 1/5000 or goat anti-mouse IgG, 1:5000, Cell Signaling Tech, USA) for 1 h at room temperature. Blots were developed using a chemiluminescence kit (Pierce, USA) and exposed to X-ray film. The bands on the film were scanned and analyzed with Quality One software (Bio-Rad). Primary antibodies and conditions used to probe blots were rabbit anti-FAK (1:1000; Cell Signaling, USA), mouse anti-β-actin (1:1000; Cell Signaling, USA). Appropriate HRP-conjugated secondary anti-rabbit or anti-mouse antibodies were used (Cell Signaling, USA). The FAK protein expression level was normalized to β-actin expression on the same nitrocellulose membrane.
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9

Western Blot Analysis of Autophagy Proteins

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U87 cells were lysed in pre-cooled RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein quantification was performed using bicinchoninic acid protein concentration determination assay (Thermo Fisher Scientific, Inc.). Following incubation in a water bath for 10 min, 25 µg protein/lane were subjected to SDS-PAGE on 12% gels. The proteins were transferred to a PVDF membrane (EMD Millipore), blocked in 5% skim milk at 25˚C for 2 h and incubated with primary antibodies (all 1:1,000) against PRDX3 (cat. no. ab73349; Abcam), autophagy-related protein 9 (ATG9; cat. no. ab108338; Abcam), Ras-related protein Rab-1 (RAB1; cat. no. 13075; Cell Signaling Technology, Inc.), p62 (cat. no. ab91526; Abcam) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) overnight at 4˚C. The membranes were subsequently washed 3 times with PBS containing 5% Tween-20 and incubated with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. ab6721; Abcam) at 25˚C for 1 h. The membranes were treated with ECL reagent (EMD Millipore) to detect the expression of proteins using an automatic chemiluminescence analyzer (Tanon 5200; Tanon Science and Technology Co., Ltd.). The band gray values were read using the Tanon GIS 4.2 software (Tanon Science and Technology Co., Ltd.).
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10

Western Blot Analysis of SNF2H Protein

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Total tissue and cell proteins were obtained using radioimmunoprecipitation assay buffer lysis buffer with proteinase inhibitors (protease inhibitor cocktail; Sigma-Aldrich, St. Louis, MO, USA). Equivalent amounts of proteins (30 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequent to blocking with 5% skimmed milk for 30 min, the membranes were incubated with a rabbit polyclonal anti-SNF2H primary antibody (dilution, 1:500; catalog no., ab3749; Abcam, Cambridge, MA, USA) at 4°C overnight. A goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (dilution, 1:3,000; catalog no., 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) was used for the subsequent incubation at room temperature for 1 h. In addition, a mouse monoclonal β-actin horseradish peroxidase-conjugated antibody (dilution, 1:5,000; catalog no., ab20272; Abcam) was used as a loading control. The expression of SNF2H was detected on X-ray film using the ECL detection system (Pierce; Thermo Fisher Scientific, Inc.).
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