ChIP assay was performed using a SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, Beverly, MA) as described in our previous report 47 (link). In brief, GH3 tumors were fixed in 37% formaldehyde and digested with micrococcal nuclease. Sheared chromatin was immunoprecipitated with anti-HIF-1α (Genetex, San Antonio, TX) or normal rabbit IgG (control) at 4℃ overnight. Purified DNAs were analyzed by qPCR with specific primers (Table S2 ).
Anti hif 1α
Manufactured by GeneTex
Sourced in United States
Anti-HIF-1α is a laboratory reagent used for the detection and quantification of HIF-1α (Hypoxia-Inducible Factor 1-alpha) protein in biological samples. It is a primary antibody that specifically binds to HIF-1α, allowing for its identification and analysis through various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
Anti actin, by GeneTex (1 mentions)
Anti dhcr24, by Thermo Fisher Scientific (1 mentions)
Anti bax, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Thermo Fisher Scientific (1 mentions)
Anti mtor, by Thermo Fisher Scientific (1 mentions)
Anti p mtor, by Thermo Fisher Scientific (1 mentions)
Anti cyclin d1, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Skimmed milk, by Merck Group (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Can get signal solution, by Toyobo (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Complete protease inhibitors cocktail, by Roche (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using anti hif 1α
1
ChIP Assay for HIF-1α Targets
2
Western Blot Analysis of Protein Expression
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The cells were lysed on ice with 100 μl of lysis buffer and centrifuged at 13,000 g for 20 min at 4 °C. The lysate proteins were quantified using BCA Protein Assay Reagent. Each sample load comprised 30 μg total proteins per lane and was resolved by 12.5% SDS-PAGE. After being transferred onto the PVDF membranes, the membranes were blocked with 5% skimmed dried milk for 1 h and incubated overnight with primary antibodies, including anti-DHCR24, anti-AKT, anti-pAKT, anti-p-GSK3beta, anti-mTOR, and anti-p-mTOR (dilution: 1:1,000; Thermo); anti-S6K1, anti-p-p53, anti-cyclin D1, and anti-cleaved caspase 3 (dilution: 1:500; Thermo); anti-pS6K1 (dilution: 1:2,000; Thermo); anti-p53 and anti-GSK3beta (dilution: 1:200; Thermo); anti-BAX (dilution: 1:2,000; Thermo); anti-p-FoxO3A, anti-HIF-1α, and anti-actin (dilution: 1:1,500; GeneTex Inc., Irvine, CA, USA). The membrane was then washed and incubated with anti-rabbit IgG secondary antibodies followed by horseradish peroxidase. The protein expressions were visualized with an ECL kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Actin acted as the loading control.
3
Co-immunoprecipitation of PER2 and HIF-1α
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Co-IP (co-inmunoprecipitation) was performed as described in our previous report 48 (link). In brief, GH3 cells were transfected with pcDNA3.1-Per2 or pcDNA3.1-Hif-1α for 48 h, and lysed in lysis buffer (Beyotime, Shanghai, China). Lysate was incubated with anti-PER2 (Affinity, Golden, CA), anti-HIF-1α (Genetex, San Antonio, TX) or anti-normal rabbit IgG (CST, Beverly, MA) overnight, followed by incubation with protein A/G magarose beads (EpiZyme Biotech, Shanghai, China) for 4 h. Bound proteins were analyzed by Western blotting.
4
Western Blot Analysis of HIF-1α Protein
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Cells were lysed with buffer containing 20 mmol/L Tris‐HCl (pH 7.5), 150 mmol/L NaCl, 0.1% SDS, 1% Triton X‐100 and cOmplete Protease Inhibitor Cocktail (Roche). The lysate was boiled at 95°C for 5 minutes for denaturing and inactivation of the endogenous proteases. The samples were separated using SDS‐PAGE (4%‐15% Mini‐PROTEAN TGX Precast Gels; Bio‐Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Bio‐Rad). The membranes were blocked with TBS containing 5% skimmed milk and 0.1% Tween 20 (Sigma‐Aldrich). The blots were then incubated with anti‐hypoxia‐inducible factor 1‐alpha (anti‐HIF‐1α) (1:500; GeneTex, Inc., Irvine, CA, USA) or β‐actin (1:1000; Epitomics, Inc., Burlingame, CA, USA) antibodies as the primary antibody at room temperature for 2 hours or at 4°C overnight. The membranes were washed three times with TBS containing 0.1% Tween 20 (TBS‐T) and then incubated with HRP‐conjugated anti‐mouse (1:1000; R&D Systems, Minneapolis, MN, USA) or anti‐rabbit IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) as the secondary antibody. Can Get Signal solution (Toyobo, Osaka, Japan) was used to reduce background noise. After the membranes were washed with TBS‐T, proteins were visualized using ECL prime (GE Healthcare). The images were acquired using a ChemiDoc imager (Bio‐Rad).
5
Western Blot Analysis of HIF-1α
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The cells were harvested and resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% NP40; 0.25% sodium deoxycholate) containing 100 μL/mL of Complete Protease Inhibitors Cocktail (Roche Applied Science, Mannheim, Germany) and 10 μL/mL of phosphatases inhibitor (Sigma-Aldrich, Saint Louis, MS). Total protein content was determined using the DC Protein Assay Kit (BioRad Laboratories, Hercules, CA). 40 μg of protein was resolved by electrophoresis on a vertical 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were incubated with primary antibodies overnight at 4°C, washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Zymed Laboratories, Invitrogen, Carlsbad, CA, USA). Proteins were detected by using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). The antibodies used were rabbit monoclonal anti-HIF-1α (GeneTex Inc., Irvine, CA, USA) diluted at 1 : 5000 and HRP-conjugated goat anti-rabbit IgG (Zymed Laboratories., Invitrogen Co., USA) diluted at 1 : 5000. As an internal control, a rabbit anti-B-Actin (GeneTex Inc.) was included.
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