* Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own experiment using appropriate negative and positive controls.
0.05% sodium azide
Product Specific Information
PA1-184 detects HIF-1 alpha in human and mouse samples, and has been successfully used in WB, ELISA, IF/ICC, and IP applications.
The PA1-184 immunogen is a partial (A.A. 530 - 825) recombinant mouse HIF-1 alpha protein expressed in E. coli.
Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP. Involved in the axonal distribution and transport of mitochondria in neurons during hypoxia.
HIF-1 alpha Antibody (PA1-184) in WB PA1184 WB_20130813104111.jpg
Western Blot with anti-HIF-1 alpha Polyclonal Antibody (PA1-184)
Western blot analysis of HIF1-alpha was performed by loading 20ug of HeLa whole cell lysate either untreated (Lane 2) or treated with 100um deferoxamine for 16 hours (Lane 3) and 7ul of Molecular Weight Protein Ladder per well onto a 4-12% Bis-Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a anti-HIF1-alpha polyclonal antibody (Product # PA1-184) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with Clean-blot IP detection reagent (Product #21230) at a dilution of 1:2000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).
HIF-1 alpha Antibody (PA1-184) in ICC PA1184 ICC_20130813103808.jpg
Immunocytochemistry with anti-HIF-1 alpha Polyclonal Antibody (PA1-184)
Immunofluorescent analysis of HIF1 alpha (green) in untreated HeLa cells (Figure A) and HeLa cells treated with 100um Deferoxamine Mesylate for ~16 hours (Figure B). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a Anti-HIF1 alpha polyclonal antibody (Product # PA1-184) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:500 for 30 minutes at room temperature. F-actin (red) was stained with DyLight 594 Phalloidin (Product # 21836) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan Instrument at 20X magnification.
HIF-1 alpha Antibody (PA1-184) in IP PA1184 IP_20130813103949.jpg
Immunoprecipitation with anti-HIF-1 alpha Polyclonal Antibody (PA1-184)
Immunoprecipitation of HIF1-alpha was performed on HeLa cells that were either untreated (Lane 2) or treated with 100um deferoxamine for 16 hours (Lane 3). Antigen-antibody complexes were formed by incubating 750ug of HeLa whole cell lysate with 2ug of an anti-HIF1-alpha polyclonal antibody (Product # PA1-184) overnight on a rocking platform at 4°C. The immune complexes were captured on 100ul Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with reducing sample loading dye. Samples were resolved on a 4-12% Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with PA1-184 at a dilution of 1:1000, washed in TBST, and probed with Clean-blot IP detection reagent (Product #21230) at a dilution of 1:2000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).
(This product is for In Vitro experimental use only. Not for resale without express authorization.)