Why is there a difference between the theoretical MW for HIF1A and the observed MW for HIF-1 alpha?

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.

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.

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).

I would like to know, does a path exist for detection of HIF 1 in venous blood before and after revascularization of the leg?

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.

What is the molecular weight (kDa) of protein HIF 1 alpha in western blot?

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

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.

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)

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 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.

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.

(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

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!

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.

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?

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.

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?

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.

Is cross-reactivity with HIF-2 alpha tested/predicted?

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.

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?

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.

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?

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.

Could you clarify if Yeast is a reactive species for HIF1a product #NB100-105?

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.

Can HIF-1 alpha Antibody (Dylight 488), product NB100-479G, react with goat species? Does this product have preservatives in it?

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, RbThe 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#sequencesGoat 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 BoratePreservative: 0.05% Sodium Azide

What's the difference between NBP2-75977 vs NBP2-75978?

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 .

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HIF-1 alpha Antibody Pack

Name: HIF-1 alpha Antibody Pack
Brand: Novus Biologicals
SKU: NB100-905
Rating: 4.5 (2 reviews)

(4 Publications)

(2 Reviews)

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Western Blot: HIF-1 alpha Antibody Pack [NB100-905] - HIF-1 alpha induction by CoCl2 on Caki-1 cell lysate. Image from verified customer review. HIF-1 alpha Antibody (H1alpha67) [NB100-105]

Biological Strategies: Western Blot: HIF-1 alpha Antibody Pack [NB100-905] - HIF-1 alpha induction by CoCl2 on Caki-1 cell lysate. Image from verified customer review. HIF-1 alpha Antibody (H1alpha67) [NB100-105]

Genetic Strategies: Knockout Validated: HIF-1 alpha Antibody Pack [NB100-905] - HIF-1 alpha was detected in immersion fixed DFO treated Hela cells (left) but was not detected in HIF-1 knockout HeLa cells (right) ...read more

Chromatin Immunoprecipitation: HIF-1 alpha Antibody Pack [NB100-905] - ChIP-qPCR assay of HIF-1 binding to hypoxia response element (HRE) of the PKM2 gene in human MDA-MB-231 breast cancer cells treated with vehicle or ...read more

Simple Western: HIF-1 alpha Antibody Pack [NB100-905] - Simple Western lane view shows a specific band for HIF-1 alpha in 0.5 mg/ml of hypoxic HeLa lysate. This experiment was performed under reducing conditions using ...read more

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-905] - HIF-1alpha expression in PDAC and adjacent normal pancreatic tissue. PDAC with weak (intensity 1) nuclear and cytoplasmic HIF-1alpha staining (left). PDAC ...read more

Flow Cytometry: HIF-1 alpha Antibody Pack [NB100-905] - Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in multiple myeloma cells: H929 cells (0.5x10^6) were stained with Alexa ...read more

Biological Strategies: Western Blot: HIF-1 alpha Antibody Pack [NB100-905] - Effects of in vitro hypoxia on the expression of mouse HIF-1 alpha in HIF-1 alpha+/- and HIF-1 alpha+/+ astrocytes. Confluent astrocyte ...read more

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody Pack [NB100-905] - Detection of HIF-1 alpha (red dye 568) in a cultured raw mouse macrophage cell line, using NB100-131. 100X magnification. Photos courtesy ...read more

Biological Strategies: Western Blot: HIF-1 alpha Antibody Pack [NB100-905] - Normoxic pre-treatment sensitizes glioma cells to radiation after graded chronic hypoxic (GCH) exposure. The results of ...read more

Biological Strategies: Western Blot: HIF-1 alpha Antibody Pack [NB100-905] - HIF-1 alpha induction on Caki-1 cell lysate using CoCl2. Image from verified customer review. HIF-1 alpha Antibody [NB100-449]

Biological Strategies: Western Blot: HIF-1 alpha Antibody Pack [NB100-905] - Effects of CoCl2 treatment on HIF-1 alpha expression in mouse astrocytes. Western blots using nuclear proteins show that both hypoxia ...read more

Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody Pack [NB100-905] - Detection of HIF-1 alpha (red dye) in a cell cytospin from a lavage of a murine skin pouch infected with Staph Aureus, using NB100-131. ...read more

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-905] - Staining in normal mouse kidney showing (A) staining with antibody and (B) blocking peptide used at 10-fold excess concentration. Images provided by Dr Yves ...read more

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-905] - Hif1alpha expression in preimplantation embryos. Co-staining of OCT4 and Hif1alpha in inner cell mass of blastocyst (top) and negative control images without ...read more

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-905] - Immunohistochemical analysis in non-GIST STS representing negative, and score 1-3 of Carbonic Anhydrase IX/CA9, GLUT-1, HIF-1 alpha, and HIF-2 alpha/EPAS1. ...read more

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-905] - Analysis of HIF-1 alpha in paraffin-embedded mouse kidney tissue section using anti-HIF-1 alpha antibody. Image from verified customer review. HIF-1 alpha ...read more

Genetic Strategies: Knockdown Validated: HIF-1 alpha Antibody Pack [NB100-905] - Morphological alterations induced by DMOG are HIF-1alpha dependent. glEND.2 cells were stably transfected with shGFP, shHIF-1alpha ...read more

Product Details

Summary

Reactivity	Hu, Mu, Rt, CaSpecies Glossary
Applications	WB, Simple Western, Flow, ICC/IF, IHC, IP

Validated by:

Biological Strategies

Genetic Strategies

Orthogonal Strategies

Order Details

100% Guarantee on every product

HIF-1 alpha Antibody Pack Summary

Description	This pack contains 1 vial each of: NB100-654SS (0.025 mL), NB100-479SS (0.025 mL), NB100-449SS (0.025 mL), NB100-105 (0.05 mL) and NB100-131 (0.05 mL).
Immunogen	For NB 100-654: Human HIF-1 alpha amino acids 432-528. For NB 100-105: Human HIF-1 alpha amino acids 432-528. For NB 100-131: Human HIF-1 alpha amino acids 329-530. For NB 100-449: Human HIF-1 alpha amino acids 775-826. For NB 100-479: Antibody was developed against a fusion protein including amino acids 530 - 825 of the mouse HIF-1 alpha protein [Uniprot# Q61221].
Specificity	These antibodies are specific for HIF-1alpha.
Gene	HIF1A

Applications/Dilutions

Dilutions

Flow Cytometry
Immunocytochemistry/ Immunofluorescence
Immunohistochemistry
Immunohistochemistry-Frozen
Immunohistochemistry-Paraffin
Immunoprecipitation
Simple Western
Western Blot

Application Notes

See individual datasheets of components for their validated applications. Western Blot: A starting dilution of 1:1,000 is recommended with titration up or down depending upon the signal and background. Immunohistochemistry: Both positive and negative feedback have been provided on all antibodies included in this pack. A starting dilution of 1:250 is recommended with titration up or down depending upon the signal and background.

Reviewed Applications

Read 2 Reviews rated 4.5

using
NB100-905 in the following applications:

Western Blot

Publications

Read Publications using
NB100-905 in the following applications:

IF/IHC
1 publication
IP
1 publication
WB
2 publications

Reactivity Notes

See individual datasheets of components for their validated species

Packaging, Storage & Formulations

Storage

Store at 4C. Do not freeze.

Kit Components

Components

Alternate Names for HIF-1 alpha Antibody Pack

AINT
anti-HIF-1 alpha
anti-HIF1A
ARNT interacting protein
ARNT-interacting protein
Basic-helix-loop-helix-PAS protein MOP1
BHLHE78
Class E basic helix-loop-helix protein 78
HIF 1A
HIF1 alpha
HIF-1 alpha
HIF1
HIF1A
HIF-1a
HIF-1alpha
HIF-1-alpha
HIF1-alpha
hypoxia inducible factor 1 alpha subunit, hypoxia inducible factor 1 subunit alpha
hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)
hypoxia-inducible factor 1-alpha
Member of PAS protein 1
member of PAS superfamily 1
MOP1
PAS domain-containing protein 8
PASD8

Background

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

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Antibody Packs are guaranteed for 1 year from date of receipt.

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Publications for HIF-1 alpha Antibody Pack (NB100-905)(4)

We have publications tested in 2 confirmed species: Human, Mouse.

We have publications tested in 3 applications: IF/IHC, IP, WB.

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Showing Publications 1 - 4 of 4.

Publications using NB100-905	Applications	Species
Gu, M et al. Silibinin inhibits inflammatory and angiogenic attributes in photocarcinogenesis in SKH-1 hairless mice. Cancer Res 67(7):3483-91. 2007-04-01 [PMID: 17409458] (WB, Mouse)	WB	Mouse
Jeon, H et al. Quercetin activates an angiogenic pathway, hypoxia inducible factor (HIF)-1-vascular endothelial growth factor, by inhibiting HIF-prolyl hydroxylase: a structural analysis of quercetin for inhibiting HIF-prolyl hydroxylase. Mol Pharmacol 71(6):1676-84. 2007-06-01 [PMID: 17377063]
Winner M, Koong AC, Rendon BE et al. Amplification of tumor hypoxic responses by macrophage migration inhibitory factor-dependent hypoxia-inducible factor stabilization. Cancer Res 2007-01-01 [PMID: 17210698] (IP, WB, Human)	IP, WB	Human
Su, H et al. Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model. Proc Natl Acad Sci U S A 99(14):9480-5. 2002-07-09 [PMID: 12084814] (IF/IHC, Mouse)	IF/IHC	Mouse

Reviews for HIF-1 alpha Antibody Pack (NB100-905) (2) 4.52

Average Rating: 4.5

(Based on 2 reviews)

We have 2 reviews tested in 1 species: Human.

Reviews using NB100-905:

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reviewed by:
Ming Lu

Human

08/08/2014

View

Summary

Application	Western Blot
Sample Tested	Human cancer cell lines
Species	Human
Lot	G

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reviewed by:
Ming Lu

Human

08/05/2014

View

Summary

Application	Western Blot
Sample Tested	Human Pancreatic Cancer Cell line
Species	Human
Lot	C-2

Product General Protocols

Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

Video Protocols

WB Video Protocol

ICC/IF Video Protocol

FAQs for HIF-1 alpha Antibody Pack (NB100-905). (Showing 1 - 10 of 11 FAQs).

Why is there a difference between the theoretical MW for HIF1A and the observed MW for HIF-1 alpha?
- 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.
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.
- 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).
I would like to know, does a path exist for detection of HIF 1 in venous blood before and after revascularization of the leg?
- 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.
What is the molecular weight (kDa) of protein HIF 1 alpha in western blot?
- The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.
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.
- 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)
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 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.
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.
- (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
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!
- 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.
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?
- 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.
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?
- 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.
Is cross-reactivity with HIF-2 alpha tested/predicted?
- 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.

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Hypoxia-Dependent CAR Stabilizing Construct in T cells Improves Solid Tumor Targeting and Efficacy
By Victoria Osinski, PhDDespite advances in the development of cancer immunotherapies, those specifically targeting tumors still remains limited. Currently, there is great interest in utilizing chimeric antigen rece... Read full blog post.

Tired T cells: Hypoxia Drives T cell Exhaustion in the Tumor Microenvironment
By Hunter MartinezThe paradigm shifting view of the immune system being leveraged to target cancer has led to numerous therapeutic breakthroughs. One major cell group responsible for this revelation is a T cell. ... Read full blog post.

Understanding ‘Y’ in Breast Cancer: Crucial Role of DNA/RNA-binding Protein YB-1 in the Development, Pre-Invasive, and Metastatic Phases
Jamshed Arslan, Pharm D, PhD In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime.1 Despite the prevalence, cancer genesis is a mystery. The heterogeneity of cancers makes it diff... Read full blog post.

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Breast cancer stem cells survive chemotherapy through S100A10-ANXA2-SPT6 interaction that epigenetically promotes OCT4-mediated stemness
By Jamshed Arslan, Pharm D, PhDBreast cancer is the most common cancer among women that causes the greatest number of cancer-related deaths worldwide. After radiotherapy or cytotoxic chemotherapy like paclitax... Read full blog post.

mTOR Signaling and the Tumor Microenvironment
By Yoskaly Lazo-Fernandez, PhD The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that, as a member of two distinct intracellular protein complexes, mTORC1 and mTORC2, regulates protein ... Read full blog post.

Bad news for stomach cancer: BAMBI protein inhibits gastric carcinoma via TGF-beta/epithelial-mesenchymal transition signaling
By Jamshed Arslan Pharm.D. Gastric carcinoma is the second leading cause of cancer-related deaths worldwide. One of the key features of gastric carcinoma is acidosis, which promotes growth and metastasis of gastric ... Read full blog post.

Developmental regulator Daam2 promotes glial cell tumors by degrading Von Hippel-Lindau protein
By Jamshed Arslan Pharm.D. Glioblastoma is an aggressive type of cancer that forms from the star-shaped glial cells of the central nervous system, called astrocytes. Intriguingly, several genes linked to glioblasto... Read full blog post.

Stemness for Surviving Hypoxia: TGF-beta/Smad Signaling in Multiple Myeloma
By Jamshed Arslan Pharm.D. Multiple myeloma (MM) is a cancer of antibody-producing plasma cells. The bone marrow (BM) of MM patients is hypoxic, and MM cells overexpress many cancerous genes that are regulated by hy... Read full blog post.

Forecasting and Targeting a Rare Cancer with Hypoxia-Inducible Factor
By Jamshed Arslan Pharm.D. Cancers of nerve, adipose, and other soft tissues are called soft tissue sarcomas (STS). Malignant peripheral nerve sheath tumor (MPNST) is an example of a rare and hard-to-treat STS; eve... Read full blog post.

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At	=	A. thaliana	SyHa	=	Golden Syrian Hamster
All	=	All Species	Ha	=	Hamster
An	=	Animal	Hu	=	Human
ArHa	=	Armenian Hamster	I	=	Insect
Av	=	Avian	Ll	=	Llama
Bb	=	Baboon	Ma	=	Mammal
Ba	=	Bacteria	Mk	=	Monkey
Bv	=	Bovine	Mu	=	Mouse
Ca	=	Canine	Multi	=	Multi-species
Ce	=	C. Elegans	NA	=	Non-species specific
Ch	=	Chicken	Or	=	Orangutan
ChHa	=	Chinese Hamster	Pl	=	Plant
Cm	=	Cynomolgus Monkey	Pm	=	Primate
Do	=	Donkey	Po	=	Porcine
Dr	=	Drosophilia	Rb	=	Rabbit
Ec	=	E. Coli	Rt	=	Rat
Eq	=	Equine	RM	=	Rhesus Macaque
Fe	=	Feline	Sh	=	Sheep
Ft	=	Ferret	Vb	=	Vertebrate
Fi	=	Fish	Vi	=	Virus
Fu	=	Fungi	Xp	=	Xenopus
Ge	=	Gerbil	Ye	=	Yeast
GP	=	Guinea Pig	Ze	=	Zebrafish
Gt	=	Goat

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