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    hif-1-alpha-antibody-ab2185.pdf

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Cardiovascular Hypoxia HIF
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Anti-HIF-1 alpha antibody (ab2185)

  • Datasheet
  • SDS
Reviews (10)Q&A (5)References (147)

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Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody (ab2185)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody (ab2185)
  • Western blot - Anti-HIF-1 alpha antibody (ab2185)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody (ab2185)

Key features and details

  • Rabbit polyclonal to HIF-1 alpha
  • Suitable for: IHC-P, WB, ICC/IF
  • Reacts with: Rat, Human
  • Isotype: IgG

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Overview

  • Product name

    Anti-HIF-1 alpha antibody
    See all HIF-1 alpha primary antibodies
  • Description

    Rabbit polyclonal to HIF-1 alpha
  • Host species

    Rabbit
  • Tested Applications & Species

    Application Species
    ICC/IF
    Human
    IHC-P
    Human
    WB
    Rat
    See all applications and species data
  • Immunogen

    Fusion protein corresponding to Human HIF-1 alpha aa 432-528.
    Database link: Q16665

  • General notes

    Under normoxic conditions HIF-1 alpha has a short half-life. It is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% and upon stabilization under hypoxic conditions HIF-1 translocates to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880). Hypoxia can be induced with treatment using certain agents e.g. CoCl2 or DFO, etc. so proper sample preparation is critical.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.05% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Cardiovascular
    • Hypoxia
    • HIF
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • HLH / Leucine Zipper
    • HLH
    • Epigenetics and Nuclear Signaling
    • Cardiovascular/Immune
    • Hypoxia
    • HIF
    • Cancer
    • Invasion/microenvironment
    • Hypoxia
    • HIF
    • Cancer
    • Cancer Metabolism
    • Response to hypoxia
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Nucleotide metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Hypoxia
    • Metabolism
    • Types of disease
    • Cancer

Associated products

  • Assay kits

    • HIF-1 alpha Transcription Factor Assay Kit (ab133104)
  • ChIP Related Products

    • Rabbit Anti-Mouse IgG H&L (ab46540)
    • ChIP Kit (ab500)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)
  • Recombinant Protein

    • Recombinant Human HIF-1 alpha protein (ab154478)
    • Recombinant Human HIF-1 alpha protein (ab48734)
  • Related Products

    • Prestained Protein Ladder - Broad molecular weight (10-245 kDa) (ab116028)
    • Hela-Vehicle treated (Negative Control) Whole Cell Lysate (ab116321)
    • Human HIF-1 alpha ELISA Kit (ab171577)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab2185 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
ICC/IF
Human
IHC-P
Human
WB
Rat
Application Abreviews Notes
IHC-P (2)
1/100 - 1/500.
WB (3)
1/500 - 1/1000.

Ensure cell lysis occurs quickly if removed from hypoxia. Ultrasonic lysis is recommended to enrich more nuclear lysate.

Positive Control: Hypoxic samples such as HeLa-DFO treated whole cell lysate ab116322. For a stronger signal, HeLa-DFO treated nuclear extracts are recommended ab180880. The cell fractionation kit can also be purchased separately ab109719. Negative Control: Most normal tissues or cells, other than kidney or heart.

The observed band size of HIF-1 alpha is not exactly as predicted due to the different forms of HIF-1 alpha below:
40-80 kDa - degraded; 10-130 kDa - post translationally modified; ~200 kDa - heterodimer with HIF-1 beta

ICC/IF (2)
1/100 - 1/500.
Notes
IHC-P
1/100 - 1/500.
WB
1/500 - 1/1000.

Ensure cell lysis occurs quickly if removed from hypoxia. Ultrasonic lysis is recommended to enrich more nuclear lysate.

Positive Control: Hypoxic samples such as HeLa-DFO treated whole cell lysate ab116322. For a stronger signal, HeLa-DFO treated nuclear extracts are recommended ab180880. The cell fractionation kit can also be purchased separately ab109719. Negative Control: Most normal tissues or cells, other than kidney or heart.

The observed band size of HIF-1 alpha is not exactly as predicted due to the different forms of HIF-1 alpha below:
40-80 kDa - degraded; 10-130 kDa - post translationally modified; ~200 kDa - heterodimer with HIF-1 beta

ICC/IF
1/100 - 1/500.

Target

  • Function

    Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions activates the transcription of over 40 genes, including, erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP.
  • Tissue specificity

    Expressed in most tissues with highest levels in kidney and heart. Overexpressed in the majority of common human cancers and their metastases, due to the presence of intratumoral hypoxia and as a result of mutations in genes encoding oncoproteins and tumor suppressors.
  • Sequence similarities

    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Domain

    Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
  • Post-translational
    modifications

    In normoxia, is hydroxylated on Pro-402 and Pro-564 in the oxygen-dependent degradation domain (ODD) by EGLN1/PHD1 and EGLN2/PHD2. EGLN3/PHD3 has also been shown to hydroxylate Pro-564. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Deubiquitinated by USP20. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
    In normoxia, is hydroxylated on Asn-803 by HIF1AN, thus abrogating interaction with CREBBP and EP300 and preventing transcriptional activation. This hydroxylation is inhibited by the Cu/Zn-chelator, Clioquinol.
    S-nitrosylation of Cys-800 may be responsible for increased recruitment of p300 coactivator necessary for transcriptional activity of HIF-1 complex.
    Requires phosphorylation for DNA-binding.
    Sumoylated; by SUMO1 under hypoxia. Sumoylation is enhanced through interaction with RWDD3. Desumoylation by SENP1 leads to increased HIF1A stability and transriptional activity.
    Ubiquitinated; in normoxia, following hydroxylation and interaction with VHL. Lys-532 appears to be the principal site of ubiquitination. Clioquinol, the Cu/Zn-chelator, inhibits ubiquitination through preventing hydroxylation at Asn-803.
    The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
  • Cellular localization

    Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
  • Target information above from: UniProt accession Q16665 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3091 Human
    • Entrez Gene: 29560 Rat
    • Omim: 603348 Human
    • SwissProt: Q16665 Human
    • SwissProt: O35800 Rat
    • Unigene: 597216 Human
    • Unigene: 10852 Rat
    • Alternative names

      • ARNT interacting protein antibody
      • ARNT-interacting protein antibody
      • Basic helix loop helix PAS protein MOP1 antibody
      • Basic-helix-loop-helix-PAS protein MOP1 antibody
      • bHLHe78 antibody
      • Class E basic helix-loop-helix protein 78 antibody
      • HIF 1A antibody
      • HIF 1alpha antibody
      • HIF-1-alpha antibody
      • HIF-1alpha antibody
      • HIF-alpha antibody
      • HIF1 A antibody
      • HIF1 Alpha antibody
      • HIF1 antibody
      • HIF1-alpha antibody
      • HIF1A antibody
      • HIF1A_HUMAN antibody
      • hifla antibody
      • Hypoxia inducible factor 1 alpha antibody
      • Hypoxia inducible factor 1 alpha isoform I.3 antibody
      • Hypoxia inducible factor 1 alpha subunit antibody
      • Hypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor antibody
      • Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor) antibody
      • Hypoxia inducible factor1alpha antibody
      • Hypoxia-inducible factor 1-alpha antibody
      • Member of PAS protein 1 antibody
      • Member of PAS superfamily 1 antibody
      • Member of the PAS Superfamily 1 antibody
      • MOP 1 antibody
      • MOP1 antibody
      • PAS domain-containing protein 8 antibody
      • PASD 8 antibody
      • PASD8 antibody
      see all

    Images

    • Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody (ab2185)
      Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody (ab2185)

      Immunocytochemical analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling HIF-1 alpha with ab2185. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2185, 1/1000 dilution) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody (ab2185)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody (ab2185)

      Immunohistochemical analysis of AN3CA (human endometrium adenocarcinoma cell line) xenograft tissue labeling HIF-1 alpha with ab2185 at 1/300 dilution. The signal was developed using HRP-labelled secondary antibody and DAB reagent, and the section was further counterstained using hematoxylin. The tested section depicted mainly a diffused cytoplasmic staining but there were some cells which showed nuclear signal also (representing hypoxic cells).

    • Western blot - Anti-HIF-1 alpha antibody (ab2185)
      Western blot - Anti-HIF-1 alpha antibody (ab2185)
      All lanes : Anti-HIF-1 alpha antibody (ab2185)

      Lane 1 : Rat nuclear extract lysate - normoxic
      Lane 2 : Rat nuclear extract lysate - hypoxic
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody (ab2185)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody (ab2185)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human kidney tissue sections labeling HIF-1-alpha with ab2185.

    Protocols

    • Nuclear Extract Preparation for HIF 1 alpha

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (147)

    Publishing research using ab2185? Please let us know so that we can cite the reference in this datasheet.

    ab2185 has been referenced in 147 publications.

    • Chen X  et al. Copper promotes the migration of bone marrow mesenchymal stem cells via Rnd3-dependent cytoskeleton remodeling. J Cell Physiol 235:221-231 (2020). PubMed: 31187497
    • Xie S  et al. Andrographolide Protects Against Adverse Cardiac Remodeling After Myocardial Infarction through Enhancing Nrf2 Signaling Pathway. Int J Biol Sci 16:12-26 (2020). PubMed: 31892842
    • Han X  et al. Advanced glycation end products enhance macrophage polarization to the M1 phenotype via the HIF-1a/PDK4 pathway. Mol Cell Endocrinol 514:110878 (2020). PubMed: 32464167
    • Kwon H  et al. Hypoxia-Preconditioned Placenta-Derived Mesenchymal Stem Cells Rescue Optic Nerve Axons Via Differential Roles of Vascular Endothelial Growth Factor in an Optic Nerve Compression Animal Model. Mol Neurobiol N/A:N/A (2020). PubMed: 32524519
    • Li J  et al. Spatial heterogeneity of oxygenation and haemodynamics in breast cancer resolved in vivo by conical multispectral optoacoustic mesoscopy. Light Sci Appl 9:57 (2020). PubMed: 32337021
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 15 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HIF-1 alpha antibody - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HEK293)
    Permeabilization
    Yes - 0.1% Triton X100
    Specification
    HEK293
    Blocking step
    Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 18 2017

    Western blot abreview for Anti-HIF-1 alpha antibody - ChIP Grade

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Notothenia coriiceps Cell lysate - other (heart)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    50 µg
    Treatment
    CTmax
    Specification
    heart
    Blocking step
    Milk as blocking agent for 10 hour(s) and 0 minute(s) · Concentration: 0.1% · Temperature: 4°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 13 2017

    Western blot abreview for Anti-HIF-1 alpha antibody - ChIP Grade

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Chaenocephalus aceratus Cell lysate - other (heart)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    50 µg
    Treatment
    CTmax
    Specification
    heart
    Blocking step
    BSA as blocking agent for 10 hour(s) and 0 minute(s) · Concentration: 0.1% · Temperature: 4°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 13 2017

    Question

    Do you recommend using nuclear lysates in case of no band?

    Read More

    Abcam community

    Verified customer

    Asked on Jul 28 2016

    Answer

    The protein HIF-1alpha does translocate to nucleus under hypoxic conditions, therefore it will be worth to try nuclear lysates as well.

    Read More

    Padamjeet Singh

    Abcam Scientific Support

    Answered on Jul 28 2016

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HIF-1-alpha antibody - ChIP Grade

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (necrotic melanoma xenograft)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9
    Permeabilization
    No
    Specification
    necrotic melanoma xenograft
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: 20°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 18 2015

    Immunocytochemistry

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
    Sample
    Mouse Cultured Cells (Neuro2A- neuroblastoma)
    Specification
    Neuro2A- neuroblastoma
    Permeabilization
    Yes - 0.3% Triton X-100
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    Submitted Jun 04 2013

    Western blot abreview for Anti-HIF1 alpha antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (Striatum (Brain))
    Loading amount
    50 µg
    Specification
    Striatum (Brain)
    Gel Running Conditions
    Reduced Denaturing (8%)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Feb 14 2013

    Question

    Hi Abcam,



    The customer is looking at these primary antibodies for HO-1 & Hif1alpha: Ab13243, ab13248, ab2185 and ab81634 to use in rat samples and in WB



    Could you please help me with their enquiry;




    Secondary antibodies, they are all goat anti rabbit, HRP or AP, can you recommend one HRP for both HO-1 and HIF-1alpha?


    ab97197, ab97200, ab97051 and ab97200




    Positive controls for HO-1 and Hif-1alpha





    MW markers suitable for HO-1 and HIF-1alpha




    I don’t understand why the predicted molecular weights of HIF-1alpha are different. Ab2185 gives 120 kDa and ab81634 gives 93 kDa.



    Can you figure it out I am a novice for WB and need help with it.



    I appreciate all your help with this,



    Kind regards,

    Read More

    Abcam community

    Verified customer

    Asked on Mar 20 2012

    Answer

    Thank you for contacting us.

    I have had a look at the four antibodies that you are interested in and there are a few points to note. The anti-Heme Oxygenase 1, ab13248, is a mouse monoclonal antibody and could therefore not be used in conjunction with any of the secondary antibodies suggested. If you would like to use this antibody in your experiments I would suggest using a secondary antibody such as https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Mouse-IgG1-heavy-chain-HRP-ab97240.htmlwhich is a goat polyclonal antibody to mouse IgG1 heavy chain.

    The secondary antibodies suggested (ab97197, ab97200, ab97051 and ab97200) would all be suitable for use in the application you wish to use them for in conjunction with the anti-Heme Oxygenase 1 antibody ab13248 and anti-HIF1 alpha ab2185. The anti-HIF1 alpha antibody, ab81634, is already HRP conjugated and therefore would not need a secondary antibody for detection.

    As positive controls,forHeme Oxygenase 1, it seems the protein is highly expressed in liver tissue which would be suitable as a control, alternatively, a cell line such as MCF-1 has also shown strong expression orHEK293 treated withhemin. For HIF-1alpha, several different celllines would be suitable such asHeLa,COS7,MCF-7,PC12, 293, or MEFcells with HIF-1alphabeing induced byeither culturing under hypoxia or using a hypoxia mimetic such as CoCl2 or DFO.

    As the proteins are expected as ˜32 kDa and 96-120 kDa, as long as the molecular weight marker covers this region it should be suitable. A marker such as https://www.abcam.com/Prism-Ultra-Protein-Ladder-10-180-kDa-ab116027.htmlor https://www.abcam.com/Prism-Protein-Ladder-10-175-kDa-ab115832.htmlwould be suitable. If you need gels, we have a new range of products available which may be of interest to you:

    https://www.abcam.com/optiblot

    These gels provide several advantages, one year shelf life and reduced batch to batch variability as well as being up to 10x stronger than conventional lab prepared or commercially available gels. Please do bear in mind that these gels use a unique polymerisation andOptiblot gels therefore need to be used with specific buffers for optimal running and transfer conditions. These can be purchased independently (https://www.abcam.com/index.html?pageconfig=resource&rid=14720), or the recepies are available on our website.

    HIF-1alpha is detected both at 93 kDa as well as 120 kDa depending on the conditions of expression. The protein sequence of HIF-1 alpha is 92.6 kDa (SwissProt reference Q16665), however, this protein then becomes post-translationally modified which increases its weight to ˜120 kDa.

    I hope this information has been of help. If you require any further information please do not hesitate to contact us again.

    Read More

    Abcam Scientific Support

    Answered on Mar 20 2012

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HIF1 alpha antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (sarcoma (MFH) subcutaneous xenografts)
    Specification
    sarcoma (MFH) subcutaneous xenografts
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.1% Triton-X100 in PBS
    Blocking step
    Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Tatiana Karakasheva

    Verified customer

    Submitted Jan 11 2012

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HIF1 alpha antibody - ChIP Grade

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (sarcoma (MFH) subcutaneous xenografts)
    Specification
    sarcoma (MFH) subcutaneous xenografts
    Fixative
    Paraformaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: citric acid
    Permeabilization
    Yes - 0.1% Triton-X100 in PBS
    Blocking step
    Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Tatiana Karakasheva

    Verified customer

    Submitted Jan 11 2012

    1-10 of 15 Abreviews or Q&A

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