Tissue lysates were prepared from the scraped colonic mucosa using RIPA lysis buffer (Cell Signaling, Danvers, MA) supplemented with protease inhibitor mixture (Roche, Indianapolis, IN). Lysates were run on a 7.5% gel and then transferred onto nitrocellulose membrane. (1×) PBS and 5% nonfat dry milk was used as a blocking buffer for 1 h. The membranes were then probed with human anti-DRA (1:100 dilution) or GAPDH antibodies (1:3000 dilution) in 1× PBS and 2.5% nonfat dry milk overnight at 4 °C. The membranes were then washed four times with wash buffer containing 1× PBS and 0.1% Tween-20 for 5 min. Finally, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2000 dilution) for 1 h and the bands were visualized with enhanced chemiluminiscence (ECL) detection reagents (Biorad, Hercules, CA). Images were acquired by imagelab 5.0 (Biorad, Hercules, CA). Quantification of band intensities was done using Image-J software.
Horseradish peroxidase conjugated goat anti rabbit igg secondary antibody
Manufactured by Bio-Rad
Sourced in United States
Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody is a laboratory reagent used in immunoassay techniques. It consists of a secondary antibody that binds to rabbit primary antibodies, with a horseradish peroxidase enzyme attached for signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Image lab 5, by Bio-Rad (1 mentions)
Ecl detection reagent, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Ecl plus reagent, by Cytiva (1 mentions)
Arginase 1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Western lightning plus ecl substrate, by PerkinElmer (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Amersham imager 600 system, by GE Healthcare (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using horseradish peroxidase conjugated goat anti rabbit igg secondary antibody
1
Western Blot Analysis of Colonic DRA
2
Quantitative Analysis of Arginase Isoforms
3
Immunoblotting Analysis of WUS Samples
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Three pairs of individual WUS samples from the same set used in the ELISA analysis were randomly selected and used for immunoblotting analysis. The protein concentration was determined using the BCA™ protein assay kit (Pierce, Rockville, IL, USA), according to the manufacturer’s protocol. Five μg of total protein in each individual sample was separated by 8% or 12% SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane and subsequently probed with one of the following antibodies: polyclonal rabbit anti-human C3c complement antibody (DAKO, Glostrup, Denmark); polyclonal goat anti-fibrinogen β antibody (Santa Cruz Biotechnology, Inc.USA,); monoclonal mouse anti-cystatin SA antibody (Abcam, Cambridge, UK). Subsequently, the membranes were incubated with one of the following antibodies: for C3c, horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Bio-Rad Laboratories, Inc.USA ,); for fibrinogen β, horseradish peroxidase-conjugated donkey anti-goat IgG secondary antibody (Bio-Rad Laboratories, Inc., USA); for cystatin SA, horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, Inc., USA). Immunoreactive protein bands were visualized using Western Lightning® Plus-ECL substrate (PerkinElmer, Inc., Waltham, MA, USA).
4
Western Blot Analysis of Cardiac Proteins
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Protein was extracted from the heart tissue by homogenizing samples in ice-cold RIPA buffer containing 50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% sodium deoxycholate, phosphatase inhibitor cocktail (Sigma-Aldrich), and a protease inhibitor cocktail (Sigma-Aldrich), followed by two cycles of centrifugation at 10,000 g for 10 min at 4 The membrane was blocked with 5% non-fat dry milk in phosphate buffered saline and 0.1% Tween-20 (PBST) for 1 h at room temperature and then incubated overnight at 4 • C with primary antibodies raised against the following: phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (Thr183/Tyr185) (p-JNK), total JNK, Cx43 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all 1:1000 dilution, from Cell Signaling Technology, Danvers, MA, USA).
The membranes were then incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG secondary antibody (all 1:5000 dilution, from Bio-Rad, Hercules, CA, USA) for 2 h at room temperature and finally visualized using a chemiluminescence ECL detection system (Millipore, Billerica, MA, USA). Images were captured using an Amersham Imager 600 system (GE Healthcare, Little Chalfont, UK).
The processing and quantification of protein bands densities were
The membranes were then incubated with horseradish peroxidaseconjugated goat anti-rabbit IgG secondary antibody (all 1:5000 dilution, from Bio-Rad, Hercules, CA, USA) for 2 h at room temperature and finally visualized using a chemiluminescence ECL detection system (Millipore, Billerica, MA, USA). Images were captured using an Amersham Imager 600 system (GE Healthcare, Little Chalfont, UK).
The processing and quantification of protein bands densities were
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