Product Pathways - Protein Translation
PathScan® Hydroxy-HIF-1α (Pro564) Sandwich ELISA Kit #13201
|13201S||1 Kit (96 assays)||---||In Stock||---|
|13201||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at firstname.lastname@example.org.
Already purchased this product? Write a Review.
|Kit Includes||Volume||Solution Color|
|HIF-1α Goat Antibody Coated Microwells||96 tests|
|Hydroxy-HIF-1α (Pro 564) Rabbit mAb Detection Antibody||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||100 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Yellowish|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
The PathScan® Hydroxy-HIF-1α (Pro564) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of hydroxy-HIF-1α (Pro564) protein. A HIF-1α antibody has been coated onto the microwells. After incubation with cell lysates, HIF-1α protein is captured by the coated antibody. Following extensive washing, a Hydroxy-HIF-1α (Pro564) Detection Antibody is added to detect the captured hydroxy-HIF-1α (Pro564) protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of hydroxy-HIF-1α (Pro564) protein.
Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
PathScan® Hydroxy-HIF-1α (Pro564) Sandwich ELISA Kit recognizes endogenous levels of hydroxy-HIF-1α (Pro564) protein, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Figure 1: Treatment of HeLa cells with the hydroxylase inhibitor dimethyloxaloylglycine (DMOG) results in decreased hydroxylation of HIF-1α, as detected by the PathScan® Hydroxy-HIF-1α (Pro564) Sandwich ELISA Kit #13201, but does not affect the level of total HIF-1α detected by PathScan® Total HIF-1α Sandwich ELISA Kit #13127. Absorbance at 450 nm is shown in the top figures while corresponding western blots using a total HIF-1α antibody (left) and Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb #3434 (right) are shown in the bottom figures. Treatment of HeLa cells with the proteasome inhibitor MG-132 #2194 stabilizes HIF-1α protein.
Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that plays a critical role in the cellular response to hypoxia (1). The HIF1 complex consists of two subunits, HIF-1α and HIF-1β, which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family (2). HIF1 regulates the transcription of a broad range of genes that facilitate responses to the hypoxic environment, including genes regulating angiogenesis, erythropoiesis, cell cycle, metabolism, and apoptosis. The widely expressed HIF-1α is typically degraded rapidly in normoxic cells by the ubiquitin/proteasomal pathway. Under normoxic conditions, HIF-1α is proline hydroxylated leading to a conformational change that promotes binding to the von Hippel Lindau protein (VLH) E3 ligase complex; ubiquitination and proteasomal degradation follows (3,4). Both hypoxic conditions and chemical hydroxylase inhibitors (such as desferrioxamine and cobalt) inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α can be induced in an oxygen-independent manner by various cytokines through the PI3K-AKT-mTOR pathway (5-7).
HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotics metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).
- Sharp, F.R. and Bernaudin, M. (2004) Nat Rev Neurosci 5, 437-48.
- Wang, G.L. et al. (1995) Proc Natl Acad Sci U S A 92, 5510-4.
- Jaakkola, P. et al. (2001) Science 292, 468-72.
- Maxwell, P.H. et al. (1999) Nature 399, 271-5.
- Fukuda, R. et al. (2002) J Biol Chem 277, 38205-11.
- Jiang, B.H. et al. (2001) Cell Growth Differ 12, 363-9.
- Laughner, E. et al. (2001) Mol Cell Biol 21, 3995-4004.
- Walisser, J.A. et al. (2004) Proc Natl Acad Sci U S A 101, 16677-82.
- Salomon-Nguyen, F. et al. (2000) Proc Natl Acad Sci U S A 97, 6757-62.
- Gunton, J.E. et al. (2005) Cell 122, 337-49.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.