Mouse anti hif 1α monoclonal antibody

Manufactured by Abcam

Sourced in United Kingdom, United States

The Mouse anti-HIF-1α monoclonal antibody is a laboratory reagent used to detect and measure the expression of the hypoxia-inducible factor 1-alpha (HIF-1α) protein in biological samples. HIF-1α is a transcription factor that plays a crucial role in the cellular response to low oxygen levels.

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Lab products found in correlation

Chemiluminescence kit, by Merck Group (1 mentions) Horseradish peroxidase hrp linked anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Rabbit anti mouse secondary antibody, by Abcam (1 mentions) Mirna qrt pcr detection kit, by GeneCopoeia (1 mentions) Glucose assay kit, by Abcam (1 mentions) Ldh activity assay kit, by Abcam (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Lactate assay kit, by Abcam (1 mentions) Atp assay kit, by Beyotime (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Rabbit anti glut1 polyclonal antibody, by Abcam (1 mentions) Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Las 3000, by Fujifilm (1 mentions) 2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)

6 protocols using mouse anti hif 1α monoclonal antibody

Immunohistochemical Detection of HIF-1α

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MCTS were harvested and fixed in cold methanol for 10 minutes at −20°C, washed in PBS, transferred to molds, embedded in OCT, and stored at −80°C until sectioning. Serial frozen sections were cut at 10μm with a cryostat, and mounted onto Superfrost Plus microscope glass slides (Menzel-Glaeser, Braunschweig, Germany). Sections were stored at −20°C until further use. Hematoxylin and eosin (H&E) staining of cryostat-sectioned slides was performed by an automatic staining workstation Tissue Stainer COT 20 (Medite GmbH, Burgdorf, Germany) with standard procedures.
For HIF-1α immunodetection, following blocking with PBS 1% goat serum for 30 minutes, sections were incubated with a mouse monoclonal anti-HIF-1α antibody (Abcam, 1:25) for 16 hours at 4°C in a dark wet chamber. Sections were then washed with bi-distilled water and incubated for 30 minutes at room temperature (RT) with a polymeric secondary antibody conjugated to alkaline phosphatase (AP Histofine Simplestain M). The primary-secondary complex was detected by enzymatic reaction using an appropriate chromogenic substrate (Histofine 415161F, New Fuchsin Substrate Kit) for 5 minutes at RT.

Däster S., Amatruda N., Calabrese D., Ivanek R., Turrini E., Droeser R.A., Zajac P., Fimognari C., Spagnoli G.C., Iezzi G., Mele V, & Muraro M.G. (2016). Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment. Oncotarget, 8(1), 1725-1736.

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Western Blot Analysis of Retinal HIF-1α

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We processed the Western blotting of retinas for the HIF-1α protein expression analysis. Fifty μg of protein per well was loaded on 8% SDS-polyacrylamide electrophoresis gel (SDS-PAGE) (P0012A, Beyotime, Beijing, China), followed by electro-transference to Polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, U.S.A) for 2 hours at 200 mA. The membranes were blocked with 5% bovine serum albumin (BSA) (A3808, MultiSciences Biotech Co., Hangzhou, China) in tris-buffered saline with tween (TBST) for 2 hours and incubated with mouse monoclonal anti-HIF-1α antibody (1:1,500; Abcam, New Territories, Hong Kong) at 4°C overnight. After being washed with TBST, the membranes were incubated with a horseradish-peroxidase-(HRP)-linked anti-mouse IgG secondary antibody (1:6,000; Cell Signaling Technology, MA, U.S.A.) for 1 hour at room temperature and washed with TBST. β-actin (1:5,000; Cell Signaling Technology, MA, U.S.A.) was used as an internal control. The bands were detected by using a chemiluminescence kit (Millipore, Billerica, MA, U.S.A) and the bands' intensities were quantitatively analyzed using Image J 1.43 U software (Wayne Rasband, National Institute of Health, U.S.A.).

Yang W., Yuan Y., Zong Y., Huang Z., Mai S., Li Y., Qian X., Liu Y, & Gao Q. (2014). Preliminary Study on Retinal Vascular and Oxygen-related Changes after Long-term Silicone Oil and Foldable Capsular Vitreous Body Tamponade. Scientific Reports, 4, 5272.

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Regulation of Glucose Metabolism by miR-33a-5p

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Glucose-Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Trizol reagent, miRNA reverse transcription kit, and Lipofectamine 2000 were purchased from Life Technologies (Waltham, MA). BCA protein assay kits were purchased from Beyotime (Haimen, China). Pre-miR-33a-5p lentivirus plasmid, anti-miR-5p lentivirus plasmid, HIF-1α overexpressing plasmid, and HIF-1α shRNA plasmid were purchased from DingGuoChangShengBiotech (Beijing, China). Sequences of the insert in plasmids were listed in Supplementary Table 1. miRNA qRT-PCR Detection Kit was purchased from GeneCopoeia, Rockville, MD, USA. RevertAid^™ H Minus First Strand cDNA Synthesis Kit was purchased from Fermentas. MTT was purchased from Biosharp (Hefei, Anhui, China). The Power SYBR Green kit was purchased from TOYOBO (Osaka, Japan). The glucose assay kit, lactate assay kit, and LDH activity assay kit were from Biovision (Milpitas, CA). The ATP assay Kit was from Beyotime Biotechnology (Shanghai). Mouse anti-HIF-1α monoclonal antibody, mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody, and rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK) and anti-lactate dehydrogenase A (LDH-A) antibody was from Epitomics (Burlingame, CA).

Cao K., Li J., Chen J., Qian L., Wang A., Chen X., Xiong W., Tang J., Tang S., Chen Y., Chen Y., Cheng Y, & Zhou J. (2017). microRNA-33a-5p increases radiosensitivity by inhibiting glycolysis in melanoma. Oncotarget, 8(48), 83660-83672.

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Cartilage Protein Expression Analysis

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Total lysates of full-thickness human cartilage were prepared in PRO-PREP buffer (iNtRON, Seoul, Korea). Protein concentration of the solutions was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Twenty micrograms of the cartilage protein and β-actin as a loading control were separated on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were soaked in 5% non-fat dried milk in TBST (10 mM/L Tris/HCl pH 7.5, 150 mM NaCl and 0.05% Tween-20) for 1 hr and then incubated for 16-18 hr with primary antibodies against HIF-1α, GLUT-1, and β-actin at 4℃. After washing three times with TBST for 10 min, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The membranes were then rinsed three times with TBST for 10 min and antigen-antibody complexes were detected using an enhanced chemiluminescence detection system (LAS-3,000; Fujifilm, Tokyo, Japan). The results were analyzed with ImageJ software comparing the density of bands. Mouse anti-HIF-1α monoclonal antibody and Rabbit anti-GLUT-1 polyclonal antibody were purchased from Abcam (Cambridge, MA, USA). Mouse anti-β-actin monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, CA, USA).

Kim M.J., Kim H.J., Hong Y.H., Lee C.K., Kim Y.W., Shon O.J, & Song I.H. (2015). Age-related NADPH Oxidase (arNOX) Activity Correlated with Cartilage Degradation and Bony Changes in Age-related Osteoarthritis. Journal of Korean Medical Science, 30(9), 1246-1252.

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Co-immunoprecipitation of AhR and HIF-1α

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A549 cells were lysed in TNE buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol). The lysates were immunoprecipitated with anti-HIF-1β/ARNT antibody (D28F3, Cell Signaling Technology) (1:50) followed by western blotting. Co-immunoprecipitation was conducted to examine the interaction of AhR and HIF-1α using the Pierce® Classic IP Kit (Rockford, IL, USA). Western blotting was performed as described above using mouse anti-HIF-1α monoclonal antibody (Abcam) (1:500) or mouse anti-AhR monoclonal antibody (Abcam) (1:500). Monoclonal mouse anti-β-actin primary antibody was used as an internal control.

Zhang M., Hu Y., Yang F., Zhang J., Zhang J., Yu W., Wang M., Lv X., Li J., Bai T, & Chang F. (2022). Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1β. BMC Pharmacology & Toxicology, 23, 26.

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Evaluating Tumor Hypoxia and Vascularization

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Nude mice bearing 4T1 tumors were i.v. injected with HSA or HSA-PTX (200 μg of PTX, 3 mg of HSA). At 24 h post injection, mice were then i.v. injected with pimonidazole hydrochloride (60 mg/kg) (Hypoxyprobe-1 plus kit, Hypoxyprobe Inc). 90 min later, tumors on those mice were then surgically excised for frozen sections. The tumor slices were incubated with mouse anti-pimonidazole antibody (dilution 1:200, Hypoxyprobe Inc.) and Alex 488-conjugated goat anti-mouse secondary antibody (dilution 1:200, Jackson Inc.) following the kits' instructions. The blood vessels were stained by incubation with rat anti-CD31 mouse monoclonal antibody (dilution 1:200, Biolegend) and Rhodamine-conjugated donkey anti-rat secondary antibody (dilution 1:200, Jackson). Cell nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (dilution 1:5000, Invitrogen). The images were captured with a confocal fluorescence microscopy (Leica SP5). For HIF-1α staining, mouse anti-HIF-1α monoclonal antibody (Abcam113642, 200 times dilution) was used as the primary antibody, while goat-anti-mouse IgG antibody conjugated with FITC (Biolegend) was used as the secondary antibody.

Tian L., Chen Q., Yi X., Wang G., Chen J., Ning P., Yang K, & Liu Z. (2017). Radionuclide I-131 Labeled Albumin-Paclitaxel Nanoparticles for Synergistic Combined Chemo-radioisotope Therapy of Cancer. Theranostics, 7(3), 614-623.

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