Cells were lysed on ice for 30 min in TNE buffer (Tris 50 mM NaCl 150 mM-EDTA 1 mM 0.5 % Triton X100) supplemented with protease inhibitors (Complete Roche Diagnostics) and lactacystin as a specific proteasome inhibitor to preserve HIF. Lysates were centrifuged at 14,000 × g for 5 min. Similar amounts of proteins from aliquots of cleared cell lysates were boiled in SDS sample buffer and analyzed by 10 % SDS-PAGE under non-reducing conditions. Proteins from gels were electrotransferred to nitrocellulose membranes followed by immunodetection with anti-HIF1α (BD, Bedford, MA, USA), anti-HIF-2α (NOVUS, Oxon, UK) and anti-γ-tubulin (Sigma, St.Louis, MO, USA) antibodies at the dilution recommended by the manufacturer. Secondary antibodies were horseradish peroxidase conjugates from Dako (Glostrup, Denmark). Membranes were developed by chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, Rockford, IL, USA).
Horseradish peroxidase conjugates
Manufactured by Agilent Technologies
Sourced in Denmark
Horseradish peroxidase (HRP) conjugates are enzyme-labeled molecules used as detection reagents in various analytical techniques. HRP is a widely used enzyme that catalyzes the oxidation of substrates, producing a colored or luminescent signal. These conjugates can be attached to antibodies, proteins, or other biomolecules to facilitate their detection and quantification in applications such as enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti γ tubulin, by Merck Group (1 mentions)
Anti hif 2α, by Novus Biologicals (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti hif 1α, by BD (1 mentions)
Ab46540, by Abcam (1 mentions)
Ab5408, by Abcam (1 mentions)
Ab81552, by Abcam (1 mentions)
Ab15597, by Abcam (1 mentions)
Ab9110, by Abcam (1 mentions)
Ab126760, by Abcam (1 mentions)
Ab7291, by Abcam (1 mentions)
Ab9116, by Abcam (1 mentions)
2 protocols using horseradish peroxidase conjugates
1
Western Blot Analysis of HIF Proteins
2
Antibody Characterization for hBRG1 and dBRM
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The antibody used to immunoprecipitate hBRG1 was the anti-ratBRG1 rabbit polyclonal antibody raised and characterized by Östlund Farrants et al. (27 (link)). The anti-ratBRG1 was also used for IP of endogenous dBRM in S2 cells. The cross-reactivity of this antibody against dBRM was shown by Tyagi et al. (11 (link)). Western blot analysis of endogenous dBRM was performed using a rabbit antibody raised against the C-terminal part of ct-BRM by Tyagi et al. (11 (link)). The rabbit anti-SNR1 and anti-MOR antibodies were raised and characterized by Dingwall et al. (28 (link)) and Mohrmann et al. (29 (link)), respectively. We also used the following commercial antibodies from Abcam: anti-hBrm (ab15597), anti-RNAPII CTD (ab5408), anti-tubulin (ab7291), anti-hCPSF1 (ab81552), anti-hCPSF2 (ab126760), anti-HA tag (ab9110), anti-V5 tag (ab9116) and anti-IgG (ab46540). Secondary antibodies for Western blotting were horseradish peroxidase conjugates purchased from DakoCytomation.
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