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 .

HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (Clone) [mFluor Violet 450 SE] - (Asp175), (CoCl2 Treated), K75D Mutant - Azide and BSA Free

Name: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (Clone) [mFluor Violet 450 SE] - (Asp175), (CoCl2 Treated), K75D Mutant - Azide and BSA Free
Brand: Novus Biologicals
SKU: NB100-105L
Price: 199 USD
Availability: Discontinued
Rating: 5 (1 reviews)

(2 Publications)

(1 Review)

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Western Blot: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (Clone) [Alexa Fluor® 555] - (Asp175) (CoCl2 Treated) - Pre-adsorbed [NB100-105L] - test1234

Immunohistochemistry: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (H1) [Alexa Fluor® 488] - (Asp175), (CoCl2 Treated) - Pre-adsorbed [NB100-105L] - test

Flow Cytometry: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (H1) [Alexa Fluor® 488] - (Asp175), (CoCl2 Treated) - Pre-adsorbed [NB100-105L] - test

Western Blot: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (H1) [Alexa Fluor® 488] - (Asp175), (CoCl2 Treated) - Pre-adsorbed [NB100-105L] - test

Western Blot: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (H1) [Alexa Fluor® 488] - (Asp175) (CoCl2 Treated) - Pre-adsorbed [NB100-105L] - Western Blot

Western Blot: HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (H1H1) [Alexa Fluor® 488] - (Asp175), (CoCl2 Treated) - Pre-adsorbed [NB100-105L] - Test

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Product Details

Summary

Product Discontinued

Order Details

Catalog Number

NB100-105L
Availability

Product Discontinued

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HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (Clone) [mFluor Violet 450 SE] - (Asp175), (CoCl2 Treated), K75D Mutant - Azide and BSA Free Summary

Description	DRAQ7 (TM) is an ideal far-red DNA counterstain for IF/IHC, high content screening and cell-based assays. DRAQ7 (TM) has many applications and is highly compatible with existing protocols across a wide range of instrumentation platforms. Key Features of DRAQ7 (TM): Does NOT enter intact, live cells Excited by blue through red lasers DRAQ7 (TM) is NON-TOXIC in long-term culture Ideal replacement for propidium iodide (and 7-AAD) having far better spectral properties: no UV excitation and no emission overlap with PE and homologues Allows a new design of combination analysis for cell health and status Viability/integrity can be directly interchanged with DRAQ5 (TM) (DNA content) Rapid staining of dsDNA/nuclei of DEAD or permeabilised cells Combination with live cell dyes for dead/live discrimination Easy to use - no wash, no RNase needed Ideal for use with GFP & FITC labels - DRAQ7 (TM) fluoresces in the far-red region NO fluorescence enhancement upon DNA binding Low photobleaching effect Compatibility with optics of flow, laser scanning cytometers and confocal and lamp-based fluorescence microscopes Cost-effective analysis
Background	BG2 - test
Immunogen	Immunogen
Epitope	EpitopeEpitope
Modification	p Ser311, Hydroxy Thr299
Target Species	Human
Localization	Localization
Marker	Marker
Specificity	SpecificitySpecificitySpecificity
Preparation Method	Preparation Method
Details of Functionality	Details of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails of FunctionalityDetails
Source	Bovine Brain
Predicted Species	Baboon (98%), Algae (95%), Armenian Hamster (100%). Backed by our 100% Guarantee.
Selection Agent	Selection Agent
Growth Properties	Growth Properties
Content	Inhibitor Content
Standard Curve Range	Standard Curve Range
Sensitivity	Sensitivity
Assay Type	Assay Type
Inter-Assay	Inter Assay
Intra-Assay	Intra Assay
Spike Recovery	Spiking Recovery
Sample Volume	Sample Volume
Kit Type	Antibody Labeling Kit
Protein/Peptide Type	Enzyme Protein Fractions
RCL Type	Stable Cell Line
Isotype	IgG2b
Clonality	Polyclonal
Host	Pig
Gene	HIF1A
Purity	Unpurified
Endotoxin Note	Endotoxin
Innovator's Reward	Test in a species/application not listed above to receive a full credit towards a future purchase. Learn about the Innovator's Reward

Applications/Dilutions

Dilutions

Binding Activity
Cell Culture 1:1000 - 1:20000
CyTOF-ready 1x10^6
Dual RNAscope ISH-IHC Compatible
ELISA 1:100 - 1:2000
Immunocytochemistry/ Immunofluorescence 1:100
Immunohistochemistry-Frozen 1:20 - 1:50
Immunohistochemistry-Paraffin 100 ug/ml for 30 minutes at RT
Western Blot 1:500

Application Notes

Application Notes

Theoretical MW

20/70 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Growth Media

NB500-201AF405 NB500-201AF405

Agonist

Survivin Antibody - BSA Free NB500-201

Antagonist

Readout System

HIF-1 alpha Antibody (H1alpha67) [7C] NB100-105V2

Control

HIF-1 alpha Antibody (H1alpha67)
HIF-1 alpha Antibody (H1alpha67)
COS-7 Nuclear Hypoxic Induced Cell Lysate

Reviewed Applications

Read 1 Review rated 5

using
NB100-105L in the following applications:

Western Blot

Publications

Read Publications using NB100-105L.

Reactivity Notes

Species Reactivity

Packaging, Storage & Formulations

Storage	Store at 4C in the dark.
Buffer	BufferBuffer - making a test edit
Preservative	0.05% Sodium Azide
Concentration	This product is unpurified. The exact concentration of antibody is not quantifiable.
Purity	Unpurified
Reconstitution Instructions	Reconstitution instructions.Reconstitution instructions.Reconstitution instructions.

Details for Array

Type	Knockout 293T Cell
Life Stage	Post-Natal
Tissue	Blood Lymph
Protein State	Poorly Differentiated
Tissue Condition	Astrocytoma Grade I
Subcellular Fraction	DTT Treated / Untreated
Position	Ventral

Notes

Public Notes This product is provided under an intellectual property license from Life Technologies Corporation. The purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment; (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

Alternate Names for Array

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
H1alpha67
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

Test123

Limitations

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

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Publications for HIF-1 alpha Antibody (NB100-105L)(2)

Showing Publications 1 - 2 of 2.

Publications using NB100-105L	Applications	Species
Florescu C, Tartea G, Streba C Et Al. The Evaluation of Beta-2-Adrenoreceptors' Expression in Normal Peritumoral Tissue in Patients with Colorectal Adenocarcinoma. Curr Health Sci J 2018-12-25 [PMID: 30581586]
Yang TH, Yao HT, Chiang MT. Red algae (Gelidium amansii) hot-water extract ameliorates lipid metabolism in hamsters fed a high-fat diet. J Food Drug Anal 2017-10-09 [PMID: 28987370]

Review for HIF-1 alpha Antibody (NB100-105L) (1) 51

Average Rating: 5

(Based on 1 review)

We have 1 review tested in 1 species: Mouse.

Reviews using NB100-105L:

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Date

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reviewed by:
Verified Customer

Mouse

03/04/2014

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Summary

Application	Western Blot
Sample Tested	samples tested
Species	Mouse
Lot	123456

Product General Protocols

View specific protocols for HIF-1 alpha Antibody (NB100-105L):

Antibody Competition protocol for HIF-1 alpha Antibody (NB100-105L)

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 (NB100-105L). (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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HIF-1 alpha Antibody (H1alpha67)
HIF-1 alpha Antibody (H1alpha67)
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Blogs on HIF-1 alpha. Showing 1-10 of 37 blog posts - Show all blog posts.

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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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Application:	WB
Species:	Mouse

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Bioinformatics

Gene Symbol	HIF1A
Entrez	11560(Avian) 11100008(Bovine)
OMIM	5678(Human)
Uniprot	P63244() Q02223() Q16665() Q16665() Q9XTW2(C. elegans) P31944(Human) O97703(Monkey)

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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

HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (Clone) [mFluor Violet 450 SE] - (Asp175), (CoCl2 Treated), K75D Mutant - Azide and BSA Free

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HIF-1 alpha [p Ser311, Hydroxy Thr299] Antibody (Clone) [mFluor Violet 450 SE] - (Asp175), (CoCl2 Treated), K75D Mutant - Azide and BSA Free Summary

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