Immunoblot analysis was performed according to a standard protocol. Briefly, after determining protein concentrations (BCA Protein Assay Kit, Pierce, Rockford, IL), 1ug of detergent-soluble retinal proteins was separated on precast 12% SDS-polyacrylamide gels (Mini-Protein TGX, BIO-RAD, Hercules, CA) and transferred onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies for Hsp70 (1:5000; Enzo, Farmingdale, NY), Hsp90 (1:5000; Enzo), Hsf1 (1:1000; Enzo) or Hsf2 (1:1000; Enzo) at 4°C overnight. Membranes were subsequently incubated with species-specific biotinylated antibodies (1:500; Enzo) and Horseradish Peroxidase (1:400; Enzo) for 1 hour at 4°C. Immunoreactive bands were detected with ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ), and their intensities were quantified with NIH image software. Reprobing blots with a monoclonal antibody to a housekeeping beta-actin (1:10000; Sigma, St. Louis, MO) was used as an internal reference to normalize protein expression levels.
Horseradish peroxidase
Manufactured by Enzo Life Sciences
Sourced in United States
Horseradish peroxidase is an enzyme derived from the roots of the horseradish plant. It is commonly used as a labeling agent in various biochemical and immunological applications due to its ability to catalyze oxidation-reduction reactions in the presence of hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Hsp90, by Enzo Life Sciences (2 mentions)
Hsp70, by Enzo Life Sciences (2 mentions)
Species specific biotinylated antibodies, by Enzo Life Sciences (2 mentions)
Lysis buffer, by Cell Signaling Technology (2 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Hrp conjugated goat anti rabbit igg, by Enzo Life Sciences (1 mentions)
Ab41928, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Dnmt3a, by Abcam (1 mentions)
Dnmt3b, by Abcam (1 mentions)
Dnmt1, by R&D Systems (1 mentions)
5 protocols using horseradish peroxidase
1
Immunoblot Analysis of Retinal Proteins
2
Immunoblot Analysis of Retinal Proteins
3
Histological Analysis of Cartilage
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Cartilage samples were fixed with 10% neutral buffered formalin (NBF) for 24 h and decalcified using 0.5 M ethylenediaminetetraacetic acid (EDTA) solution for a week. After paraffin embedding, blocks were cut at 5 μM thickness and stained with safranin O. For immunohistochemical analysis, deparaffinized sections were incubated with primary antibodies overnight at 4 °C in a humidified chamber. Sections were developed using ImmPACT DAB (Vector Laboratories, #SK‐4105). The following antibodies were used for immunohistochemical analysis: PPARγ (1:100 dilution, Abcam, #ab41928), IL-1β (1:100 dilution, Abcam, #ab9722), Bodipy (1:50 dilution, Thermo Fisher Scientific), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:200 dilution, Enzo Life Sciences, #ADI‐SAB‐300).
4
Western Blot Protein Detection Protocol
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Cells were lysed with lysis buffer (Cell signaling technology, Danvers, MA, USA). Then, protein samples (20 μg) were separated by SDS-PAGE, and were blotted onto polyvinylidene difluoride membranes (BIO-RAD Laboratories, Hercules, CA, USA). After blocking with 1% (w/v) non-fat dry milk powder for 1 h, the membranes were incubated with primary antibodies overnight at 4°C, then with secondary antibody conjugated with horseradish peroxidase (Enzo Life Sciences, Inc., Farmingdale, NY, USA). Specific antigen-antibody complexes were detected by enhanced chemiluminescence using BioFx® Chemiluminescent Sensitive Plus HRP (SurModics. IVD. Inc., Eden Prairie, MN, USA). The specific band was visualized using Ez-Capture ST chemiluminescence imaging system (ATTO, Tokyo, Japan). Specific antibodies are presented in Supplementary Table 1 . Experiments were performed in repeated three times with similar results.
5
Western Blot Analysis of Protein Targets
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The cells were lysed in lysis buffer (cell signaling tech. Beverly, MA, USA) for 15 min on ice. The protein concentration of the lysates was measured using a Bradford assay (Bio-Rad, Hercules, CA, USA). Protein extracts (50 μg) were separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinyl difluoride membranes. After blocking with 2% skim milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C, then with secondary antibody conjugated with horseradish peroxidase (Enzo Life Sciences, Farmingdale, NY, USA) for 2 h. The membrane was visualized by Super Signal® West Pico Chemiluminescent Substrate (Thermo scientific, Rockford, IL, USA) and images were produced using medical X-ray film blue (Agfa health care NV, Belgium). Specific antibodies were as follows: p-glycoprotein antibody (Merck Millipore, Billerica, MA, USA), hENT1 (Novus Biologicals, Littleton, CO, USA), DNMT1 (R&D Systems, Minneapolis, MN, USA), DNMT3a (Abcam, Cambridge, MA, USA), DNMT3b (Abcam), and β-actin (Sigma, St. Louis, MO, USA).
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