Rat HIF-1 alpha ELISA Kit (Colorimetric)

Novus Biologicals | Catalog # NBP3-49777

Novus Biologicals
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Key Product Details

Sample Type & Volume Required Per Well

Serum, plasma and other biological fluids (100 uL)

Sensitivity

4.69 pg/mL

Assay Range

7.81 - 500 pg/mL
View all HIF-1 alpha/HIF1A ELISA Kits »

Product Specifications

Assay Type

Sandwich ELISA

Kit Type

ELISA Kit (Colorimetric)

Species

Rat

Specificity

This kit recognizes Rat HIF-1 alpha in samples. No significant cross-reactivity or interference between Rat HIF-1 alpha and analogues was observed

Description

Assay Length: 3 h 30 min

Precision

Intra-Assay Precision (Precision within an assay) CV% 10

Inter-Assay Precision (Precision between assays) CV% 10

Recovery for Rat HIF-1 alpha ELISA Kit (Colorimetric)

Recovery

80 - 120%

Linearity

Rat HIF-1 alpha ELISA Kit (Colorimetric)

Scientific Data Images for Rat HIF-1 alpha ELISA Kit (Colorimetric)

Rat HIF-1 alpha ELISA Kit (Colorimetric)

ELISA: Rat HIF-1 alpha ELISA Kit (Colorimetric) [NBP3-49777]

Standard Curve Reference.

Kit Contents for Rat HIF-1 alpha ELISA Kit (Colorimetric)

  • Biotinylated Detection Ab Diluent
  • Certificate of Analysis
  • Concentrated Biotinylated Detection Ab (100x)
  • Concentrated HRP Conjugate (100x)
  • Concentrated Wash Buffer (25x)
  • HRP Conjugate Diluent
  • Micro ELISA Plate (Dismountable)
  • Plate Sealer
  • Product Description
  • Reference Standard
  • Reference Standard & Sample Diluent
  • Stop Solution
  • Substrate Reagent

Preparation and Storage

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Storage of components varies. See protocol for specific instructions.

Background: HIF-1 alpha/HIF1A

Hypoxia contributes to the pathophysiology of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease (1). In cancer and particularly solid tumors, hypoxia plays a critical role in the regulation of genes involved in stem cell renewal, epithelial to mesenchymal transition (EMT), metastasis and angiogenesis. In the tumor microenvironment (TME), hypoxia influences the properties and function of stromal cells (e.g., fibroblasts, endothelial and immune cells) and is a strong determinant of tumor progression (2,3).

HIF-1 or hypoxia inducible factor 1 (predicted molecular weight 93kDa), is a transcription factor commonly referred to as a "master regulator of the hypoxic response" for its central role in the regulation of cellular adaptations to hypoxia. In its active form under hypoxic conditions, HIF-1 is stabilized by the formation of a heterodimer of HIF-1 alpha and ARNT/HIF-1 beta subunits. Nuclear HIF-1 engages p300/CBP for binding to hypoxic response elements (HREs). This process induces transcription and regulation of genes including EPO, VEGF, iNOS2, ANGPT1 and OCT4 (4,5).

Under normoxic conditions, the HIF-1 alpha subunit is rapidly targeted and degraded by the ubiquitin proteasome system. This process is mediated by prolyl hydroxylase domain enzymes (PHDs), which catalyze the hydroxylation of key proline residues (Pro-402 and Pro-564) within the oxygen-dependent degradation domain of HIF-1 alpha. Once hydroxylated, HIF-1 alpha binds the von Hippel-Lindau tumor suppressor protein (pVHL) for subsequent ubiquitination and proteasomal degradation (4). pVHL dependent regulation of HIF-1 alpha plays a role in normal physiology and disease states. Regulation of HIF-1 alpha by pVHL is critical for the suppressive function of FoxP3+ regulatory Tcells (6). Repression of pVHL expression in chronic lymphocytic leukemia (CLL) B cells leads to HIF-1 alpha stabilization and increased VEGF secretion (7).

References

1. Semenza, G. L., Agani, F., Feldser, D., Iyer, N., Kotch, L., Laughner, E., & Yu, A. (2000). Hypoxia, HIF-1, and the pathophysiology of common human diseases. Advances in Experimental Medicine and Biology.

2. Muz, B., de la Puente, P., Azab, F., & Azab, A. K. (2015). The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia. https://doi.org/10.2147/hp.s93413

3. Huang, Y., Lin, D., & Taniguchi, C. M. (2017). Hypoxia inducible factor (HIF) in the tumor microenvironment: friend or foe? Science China Life Sciences. https://doi.org/10.1007/s11427-017-9178-y

4. Koyasu, S., Kobayashi, M., Goto, Y., Hiraoka, M., & Harada, H. (2018). Regulatory mechanisms of hypoxia-inducible factor 1 activity: Two decades of knowledge. Cancer Science. https://doi.org/10.1111/cas.13483

5. Dengler, V. L., Galbraith, M. D., & Espinosa, J. M. (2014). Transcriptional regulation by hypoxia inducible factors. Critical Reviews in Biochemistry and Molecular Biology. https://doi.org/10.3109/10409238.2013.838205

6. Lee, J. H., Elly, C., Park, Y., & Liu, Y. C. (2015). E3Ubiquitin Ligase VHL Regulates Hypoxia-Inducible Factor-1 alpha to Maintain Regulatory T Cell Stability and Suppressive Capacity. Immunity. https://doi.org/10.1016/j.immuni.2015.05.016

7. Ghosh, A. K., Shanafelt, T. D., Cimmino, A., Taccioli, C., Volinia, S., Liu, C. G.,... Kay, N. E. (2009). Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells. Blood. https://doi.org/10.1182/blood-2008-10-185686

Long Name

Hypoxia Inducible Factor 1 Subunit Alpha

Alternate Names

BHLHE78, HIF 1A, HIF-1a, HIF1 alpha, HIF1A, MOP1, PASD8

Gene Symbol

HIF1A

Additional HIF-1 alpha/HIF1A Products

Product Documents for Rat HIF-1 alpha ELISA Kit (Colorimetric)

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Product Specific Notices for Rat HIF-1 alpha ELISA Kit (Colorimetric)

This product is for research use only and is not approved for use in humans or in clinical diagnosis. ELISA Kits are guaranteed for 6 months from date of receipt.

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FAQs for Rat HIF-1 alpha ELISA Kit (Colorimetric)

Showing  1 - 5 of 15 FAQs Showing All

    • Q: Can HIF-1 alpha Antibody (Dylight 488), product NB100-479G, react with goat species? Does this product have preservatives in it?
      A: NB100-479G has not been tested in goat species. Only the listed species on the product page and datasheet will be guaranteed. 
      Reactivity: Hu, Mu, Rt, Ca, Fi, Ha, Pm, Rb

      The immunogen for this antibody corresponds to amino acids 530-825 of mouse Hifa. Running a sequence alignment of this sequence with the goat sequence found on UniProt yields around 81% homology.Mouse Hif1a: https://www.uniprot.org/uniprot/Q61221#sequences
      Goat Hif1a: https://www.uniprot.org/uniprot/A0A023R978#sequencesLastly, there is 0.05% Sodium Azide present in the formulation of the product. This is also listed on the product page and datasheet. 
      Buffer: 50mM Sodium Borate
      Preservative: 0.05% Sodium Azide

    • A: This HIF-1 alpha antibody has not been tested in yeast. The homology is not significantly homologous so we do not believe there will be cross reactivity to the yeast protein. 

    • Q: I am curious to know the biochemical reactions of CoCl2 that mimic hypoxia. Is it that CoCl2 can bind any ubiquitin enzyme which regulates their degradation?
      A: CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

    • Q: I am doing HIF1 westerns in HIF-overexpressing mouse liver and adipose tissue using Novus antirabbit HIF1a antibody with overnight incubation. I am getting strong bands around 90kDa. I am aware that HIF theoretical molecular weight is 93kDa, but in westerns, the HIF band is usually around 120kDa according to my internet research. Can someone let me know if I’m getting the right HIF band or just some non-specific bands? Thanks.
      A: (1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.
      (2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.
      (3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.
      (4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs 
      (5)    Last but not the least, Novus technical support team may be contacted via email

    • Q: I have Hif1a nuclear protein extract at -80C. I am wondering if anyone knows how long it would be good for at that temperature since HIf1a is known to be degraded easily.
      Thank you!
      A: You could try a few things to further inhibit the degradation.
      1) Use the protease inhibitors (if you are not already using them).
      2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.
      3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).
      5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.
      6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.

    • Q: I performed several Western Blots of HIF-1 alpha with different lysis buffers, whole lysates, and cytoplasm/nuclei extractions. I can’t seem to get a good western blot (poor signal, band much lower than expected, etc.). Can someone suggest some technical considerations/tricks I should consider using?
      A: A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.
      For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.

    • Q: I would like to know, does a path exist for detection of HIF 1 in venous blood before and after revascularization of the leg? 
      A: We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.

    • A: Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.

    • Q: Our lab recently ordered NB100-449, HIF-1 alpha antibody. Unfortunately an inexperienced technician stored it at -20C rather than 4C for approximately 2 days. Have you done any tests to determine antibody functionality if frozen?
      A: The recommended storage condition of HIF-1 alpha antibody NB100-449 is 4C and we highly recommended not storing the product lower than the freezing point, as it may potentially disrupt the protein folding and destroy the antigen binding site of the antibody. Since we likely have not tested a storage condition of -20C for this antibody, we cannot really say if this antibody has been impaired by the storage condition. Our recommendation would be to test the antibody in a small portion of your treated cell line and see if the antibody is still reactive to the HIF-1 alpha protein.

    • Q: We got the Hif1a (NB100-105) antibody from you guys. I used the concentration that is mentioned on your website, but I am getting a band of a completely different size (~70kDa) and not the 120 kDa mentioned.
      A: HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.

      Please go through our hypoxia related FAQs, you should find them very informative.Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 
      1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)
      2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)

    • Q: We ordered and received the HIF-1 alpha antibody NB100-449 and on the packing slip it says that is prepared in TBS+0.1% BSA. I will be using it for western blots. DO you think my choice of blocking buffer (milk or Blotto) could interfere with the activity of this antibody or should it be necessarily BSA based?
      A: Choice of blocking buffer is entirely within your discretion; it will not affect the antibody binding activity. Please note, some blocking buffers may work better than others and sometimes optimization is needed.

      When working with hypoxia there are other important factors to consider, as HIF-alpha is very easily degraded. The lysates should be freshly prepared. Also lysate preparation should be as quick as possible to avoid any exposure to oxygen - we recommend lysing cells directly into the SDS sample loading buffer (Laemmli buffer). We also highly recommend using positive control (you can prepare them yourself or  choose some of those we have for sale).

      I have attached some additional information that you may find quite useful. Also here are some hypoxia related FAQ addressing common concerns.

    • A: The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.

    • A: While the same immunogen was used to make both HIF-1 alpha antibodies, they are different clones, meaning they recognize a different epitope on the immunogen.

    • Q: Which antibody(ies) do you recommend for the detection of HIF-1a by immunohistochemistry in the sections of paraffin-embedded mouse liver samples? I would appreciate if you can give me several choices and rank them in the order of performance. My goal is to distinguish HIF upregulation by prolyl hydroxylase inhibitor in different liver cells.
      A: All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).

    • Q: Why is there a difference between the theoretical MW for HIF1A and the observed MW for HIF-1 alpha?
      A: HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.
Showing  1 - 5 of 15 FAQs Showing All
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