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.

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).

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.

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 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.

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.

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.

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.

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.

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.

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 .

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.

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)

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, 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#sequences Lastly, 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

HIF-1 alpha/HIF1A Peptide Substrate, Biotin

Name: HIF-1 alpha/HIF1A Peptide Substrate, Biotin
Brand: R&D Systems
SKU: S-340

R&D Systems, part of Bio-Techne | Catalog # S-340

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S-340 has been discontinued. View all HIF-1 alpha/HIF1A products.

Product Specifications
Preparation & Storage
Background
Product Documents
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Key Product Details

Source

Synthetic

Accession #

Q16665

Conjugate

Biotin

Applications

Bioactivity

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Product Specifications

Source

Chemically Synthesized

Purity

>98%, by HPLC.

Predicted Molecular Mass

5290.9 Da

Formulation, Preparation and Storage

S-340

Formulation	100 μM in 10% (w/v) ammonium bicarbonate.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage	Storeat -20°C or -80°C. Avoid multiplefreeze/thaw cycles.

Background: HIF-1 alpha/HIF1A

This biotinylated peptide includes a sequence from human Hypoxia-Inducible Factor 1-alpha (HIF-1 alpha) including a 4-hydroxyline proline residue corresponding to proline 564 in UniProt Q16665. The peptide can be ubiquitinated by a VHL/Elongin B/Elongin C/CUL2/RBX complex (catalog number E3-655) in vitro using wild-type ubiquitin or lysine-less ubiquitin ("Ub-NOK"). A matched negative control peptide (catalog S-345) with an unmodified proline residue is available; depending on experimental conditions the negative control peptide is ubiquitinated at ≤ 5% of the level of the peptide containing 4-hydroxyproline.

Long Name

Hypoxia Inducible Factor 1 Subunit Alpha

Alternate Names

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

Entrez Gene IDs

3091 (Human); 15251 (Mouse); 29560 (Rat)

Gene Symbol

HIF1A

UniProt

Q16665

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Product Documents for HIF-1 alpha/HIF1A Peptide Substrate, Biotin

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Product Specific Notices for HIF-1 alpha/HIF1A Peptide Substrate, Biotin

For research use only

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HIF-1 alpha/HIF1A Peptide Substrate, Biotin

R&D Systems, part of Bio-Techne | Catalog # S-340

Key Product Details

Source

Accession #

Conjugate

Applications

View all HIF-1 alpha/HIF1A Small Molecules and Peptides »

Product Specifications

Source

Purity

Predicted Molecular Mass

Formulation, Preparation and Storage

S-340

Background: HIF-1 alpha/HIF1A

Long Name

Alternate Names

Entrez Gene IDs

Gene Symbol

UniProt

Additional HIF-1 alpha/HIF1A Products

Product Documents for HIF-1 alpha/HIF1A Peptide Substrate, Biotin

Product Datasheets

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SDS

Product Specific Notices for HIF-1 alpha/HIF1A Peptide Substrate, Biotin

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