For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online
United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Customized Products & Partnerships
    Customized Products & Partnerships

    Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs.

    Customized products

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

  1. Link

    products/primary-antibodies/hnf-4-alpha-antibody-epr3648-ab92378.pdf

  1. Send me a copy of this email
    I agree to the terms and conditions.
Cardiovascular Blood Coagulation Extrinsic
Share by email
RecombinantRabMAb

Recombinant Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

  • Datasheet
  • SDS
Reviews (4)Q&A (5)References (14)

Product price, shipping and contact information

Currently unavailable

Sorry, we can't display this right now.

Please contact us to place your order, or try again later.

 

Loading size & price…

 

Shipping and order information

Shipping info

Promotion Information

Abpromise

Guaranteed product quality, expert customer support.

Find out more.

Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • ChIP-sequencing - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
  • Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR3648] to HNF-4-alpha
  • Suitable for: WB, IHC-P, ICC/IF, Flow Cyt (Intra), ChIP-sequencing, ChIC/CUT&RUN-seq
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 647 APC Carrier Free HRP PE

You may also be interested in

Protein
Product image
Recombinant Human HNF-4-alpha protein (ab132090)
ELISA
Product image
Human PCSK9 ELISA Kit (ab209884)
Primary
Product image
Alexa Fluor® 488 Anti-HNF-4-alpha antibody [EPR3648] (ab216897)

View more associated products

Overview

  • Product name

    Anti-HNF-4-alpha antibody [EPR3648]
    See all HNF-4-alpha primary antibodies
  • Description

    Rabbit monoclonal [EPR3648] to HNF-4-alpha
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt (Intra), ChIP-sequencing, ChIC/CUT&RUN-seqmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HepG2, A549 and SW480 cell lysates. IHC-P: Human colon and kidney tissues. ICC/IF: HepG2 cells. Flow Cyt (intra): HepG2 cells. ChIC/CUT&RUN-seq: HepG2 cells. ChIP-seq: HepG2 cells.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

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: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR3648
  • Isotype

    IgG
  • Research areas

    • Cardiovascular
    • Blood
    • Coagulation
    • Extrinsic
    • Cardiovascular
    • Lipids / Lipoproteins
    • Lipid Metabolism
    • Cholesterol Metabolism
    • Cardiovascular
    • Lipids / Lipoproteins
    • Lipid Metabolism
    • Cytochromes
    • Signal Transduction
    • Metabolism
    • Energy Metabolism
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Polymerase associated factors
    • Pol II Transcription
    • Other
    • Cardiovascular
    • Atherosclerosis
    • Diabetes associated
    • Developmental Biology
    • Organogenesis
    • Gut development
    • Liver development
    • Cancer
    • Cancer Metabolism
    • Metabolic signaling pathway
    • Metabolism of lipids and lipoproteins
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Lipid metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Cholesterol Metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Lipases
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Energy transfer pathways
    • Energy Metabolism
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Cytochromes
    • Metabolism
    • Types of disease
    • Diabetes
    • Metabolism
    • Types of disease
    • Cancer
    • Metabolism
    • Types of disease
    • Heart disease

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-HNF-4-alpha antibody [EPR3648] (ab216897)
    • Alexa Fluor® 647 Anti-HNF-4-alpha antibody [EPR3648] (ab217073)
    • Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Positive Controls

    • Hep G2 nuclear extract lysate (ab14660)
    • Hep G2 whole cell lysate (ab166833)
    • SW480 whole cell lysate (ab3957)
    • A549 whole cell lysate (ab7910)
  • Recombinant Protein

    • Recombinant Human HNF-4-alpha protein (ab132090)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab92378 in the following tested applications.

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

Application Abreviews Notes
WB (1)
1/2000. Predicted molecular weight: 53 kDa.

For unpurified use at 1/1000 - 1/10000.

IHC-P (1)
1/250 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.

ICC/IF (2)
1/100 - 1/250.
Flow Cyt (Intra)
1/70.

For unpurified use at 1/100.  ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ChIP-sequencing
Use at an assay dependent concentration.
ChIC/CUT&RUN-seq
Use at an assay dependent concentration.
Notes
WB
1/2000. Predicted molecular weight: 53 kDa.

For unpurified use at 1/1000 - 1/10000.

IHC-P
1/250 - 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.

ICC/IF
1/100 - 1/250.
Flow Cyt (Intra)
1/70.

For unpurified use at 1/100.  ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ChIP-sequencing
Use at an assay dependent concentration.
ChIC/CUT&RUN-seq
Use at an assay dependent concentration.
Application notes
Is unsuitable for IP.

Target

  • Function

    Transcriptionally controlled transcription factor. Binds to DNA sites required for the transcription of alpha 1-antitrypsin, apolipoprotein CIII, transthyretin genes and HNF1-alpha. May be essential for development of the liver, kidney and intestine.
  • Involvement in disease

    Defects in HNF4A are the cause of maturity-onset diabetes of the young type 1 (MODY1) [MIM:125850]; also symbolized MODY-1. MODY is a form of diabetes that is characterized by an autosomal dominant mode of inheritance, onset in childhood or early adulthood (usually before 25 years of age), a primary defect in insulin secretion and frequent insulin-independence at the beginning of the disease.
  • Sequence similarities

    Belongs to the nuclear hormone receptor family. NR2 subfamily.
    Contains 1 nuclear receptor DNA-binding domain.
  • Post-translational
    modifications

    Phosphorylated on tyrosine residue(s); phosphorylation is important for its DNA-binding activity. Phosphorylation may directly or indirectly play a regulatory role in the subnuclear distribution.
  • Cellular localization

    Nucleus.
  • Target information above from: UniProt accession P41235 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3172 Human
    • Omim: 600281 Human
    • SwissProt: P41235 Human
    • Unigene: 116462 Human
    • Alternative names

      • FLJ39654 antibody
      • FRTS4 antibody
      • Hepatic nuclear factor 4 alpha antibody
      • Hepatocyte nuclear factor 4 alpha antibody
      • Hepatocyte nuclear factor 4 antibody
      • Hepatocyte nuclear factor 4-alpha antibody
      • HNF 4 alpha antibody
      • HNF 4 antibody
      • HNF-4-alpha antibody
      • HNF4 antibody
      • HNF4A antibody
      • HNF4A_HUMAN antibody
      • HNF4a7 antibody
      • HNF4a8 antibody
      • HNF4a9 antibody
      • Hnf4alpha antibody
      • HNF4alpha10/11/12 antibody
      • MODY 1 antibody
      • MODY antibody
      • MODY1 antibody
      • NR2A1 antibody
      • NR2A21 antibody
      • Nuclear receptor subfamily 2 group A member 1 antibody
      • OTTHUMP00000031060 antibody
      • OTTHUMP00000031062 antibody
      • TCF 14 antibody
      • TCF antibody
      • TCF-14 antibody
      • TCF14 antibody
      • Tcf4 antibody
      • Transcription factor 14, hepatic nuclear factor antibody
      • Transcription factor 14 antibody
      • Transcription factor HNF 4 antibody
      • Transcription factor HNF-4 antibody
      • Transcription factor HNF4 antibody
      see all

    Images

    • Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/2000 dilution (purified) + SW480 cell lysate at 20 µg

      Secondary
      Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 53 kDa
      Observed band size: 53 kDa



      Blocking and dilution buffer: 5% NFDM/TBST.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling HNF-4-alpha with purified ab92378 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling HNF-4 with purified ab92378 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.

      Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

      Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

    • Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/5000 dilution (purified) + HepG2 cell lysate at 10 µg

      Secondary
      Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 53 kDa
      Observed band size: 53 kDa



      Blocking and dilution buffer: 5% NFDM/TBST.

    • Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Western blot - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      All lanes : Anti-HNF-4-alpha antibody [EPR3648] (ab92378) at 1/1000 dilution (unpurified)

      Lane 1 : HepG2 cell lysate
      Lane 2 : A549 cell lysate
      Lane 3 : SW480 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

      Predicted band size: 53 kDa

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue (A) and human kidney tissue (B) labelling HNF-4-aplha with unpurified ab92378 at a 1/100 dilution. Detection: DAB staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

    • Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Immunocytochemistry/Immunfluorescence analysis of HepG2 cells labelling HNF-4-alpha with unpurified ab92378 at a 1/100 dilution.

    • Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Intracellular Flow Cytometry analysis of HepG2 cells labelling HNF-4 with purified ab92378 at 1/70 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

       

       

    • Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Overlay histogram showing HepG2 cells stained with unpurifiedab92378 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified ab92378, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

       

       

    • ChIP-sequencing - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      ChIP-sequencing - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      Chromatin was prepared from HepG2 (Human liver hepatocellular carcinoma cell line) cells. ChIP was performed with 10^7 HepG2 cells and 8 µg of ab92378 [EPR3648]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

      Additional screenshots of mapped reads can be downloaded here.

    • ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

      CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 2.5 x 105 HepG2 (Human liver hepatocellular carcinoma cell line) cells and 5 µg of ab92378 [EPR3648]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.

      Additional screenshots of mapped reads can be downloaded here.

    • Anti-HNF-4-alpha antibody [EPR3648] (ab92378)
      Anti-HNF-4-alpha antibody [EPR3648] (ab92378)

    Protocols

    • Flow cytometry protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (14)

    Publishing research using ab92378? Please let us know so that we can cite the reference in this datasheet.

    ab92378 has been referenced in 14 publications.

    • Wang N  et al. HDAC6/HNF4a loop mediated by miR-1 promotes bile acids-induced gastric intestinal metaplasia. Gastric Cancer 24:103-116 (2021). PubMed: 32705446
    • Borys BS  et al. Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors. Stem Cell Res Ther 12:55 (2021). PubMed: 33436078
    • Huang KW  et al. Liver Activation of Hepatocellular Nuclear Factor-4a by Small Activating RNA Rescues Dyslipidemia and Improves Metabolic Profile. Mol Ther Nucleic Acids 19:361-370 (2020). PubMed: 31877412
    • Famulari ES  et al. Human liver stem cells express UGT1A1 and improve phenotype of immunocompromised Crigler Najjar syndrome type I mice. Sci Rep 10:887 (2020). PubMed: 31965023
    • Rohani L  et al. Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells. Commun Biol 3:492 (2020). PubMed: 32895477
    View all Publications for this product

    Customer reviews and Q&As

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

    1-9 of 9 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HNF-4-alpha antibody [EPR3648]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Human induced pluripotent stem cell derived hepato)
    Permeabilization
    Yes - 0.5% Triton in PBS
    Specification
    Human induced pluripotent stem cell derived hepato
    Blocking step
    BSA 1% + Donkey Serum 3% + Triton 0.1 % in PBS as blocking agent for 20 minute(s) · Concentration: 1% · Temperature: 22°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Dr. Candice Ashmore-Harris

    Verified customer

    Submitted Sep 02 2020

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HNF-4-alpha antibody [EPR3648]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Kidney H9 Organoid)
    Permeabilization
    Yes - 0.05% tween 20
    Specification
    Kidney H9 Organoid
    Blocking step
    Sea Block as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 22°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Jan 27 2020

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HNF-4-alpha antibody [EPR3648]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Kidney)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Tris/EDTA buffer pH9 ab94681
    Permeabilization
    Yes - 0.05% Tween 20 in PBS
    Specification
    Kidney
    Blocking step
    Sea Block as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 22°C
    Fixative
    10%NBF
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    MR. David Ivancic

    Verified customer

    Submitted May 21 2018

    Western blot abreview for Anti-HNF-4-alpha antibody [EPR3648]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (ESC (HUES7))
    Gel Running Conditions
    Reduced Denaturing (12)
    Loading amount
    10 µg
    Specification
    ESC (HUES7)
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Aug 09 2016

    Question

    I kindly ask you to send me the protocols, information regarding pre-treatment etc. for the following abs:

    Read More

    Abcam community

    Verified customer

    Asked on Jun 21 2013

    Answer

    Our antigen retrieval method used for ab92378 and ab129189 is described below.

    Antigen Retrieval

    This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

    1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.

    2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.

    3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.

    4. Let it cool down to room temperature (10 - 20 minutes).

    5. Removes slides and rinse in TBST.

    6. Proceed to Staining step.

    Read More

    Tanya Bagrij

    Abcam Scientific Support

    Answered on Jun 21 2013

    Question

    In the specification sheet for Pax8 on your website (see in attachment) stands 100 ug and in the mail below – 250 ul. Can you please clarify this to me?

    Our customer is asking for this antibodies for IHC. Can you please send me the working procedure for the antibodies.

    Thank you in advance.

    Best regards,

    Read More

    Abcam community

    Verified customer

    Asked on Sep 27 2012

    Answer

    Thank you fo ryour message.

    To clarify regarding ab135618 Anti-PAX8 antibody, the datasheet states we sell this at 100 ug, at a concentration of 0.25 mg/ml. Therefore, you can calculate that at this concentrations, 100 ug will be in 250 ul of liquid.

    For the protocols, I am pleased to provide the general testing IHC-P protocols from the originators. I have copied thesebelow. I am sorry it has taken a while to obtain these.Please note that these will be a guideline only and further individual optimizationmay be required.

    As stated in the previous email, recommended dilutions in IHC-P for these antibodies are listed on the datasheets:

    ab129189 NAPSIN AIHC-P: 1/100 - 1/250.
    ab92378 HNF4 IHC-P: 1/100 - 1/250
    ab135618 PAX8IHC-P: 1/50 - 1/100.

    I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

    Protocol for ab135618 Anti-PAX8 antibody


    Preparing Tissue Sections for Immunostaining:

    1. Fix the tissue in 10% formalin at 4oC overnight.
    2. Embed fixed tissue in paraffin.
    3. Mount tissue sections on slides.
    4. Clear the paraffin with xylene for ten minutes; move slides to a fresh dish of xylene for an additional ten minutes. NOTE: Perform all xylene washes in a fume hood!
    5. Rinse the slides twice for 2 minutes in 100% alcohols (18:1:1 100% ethanol: 100% methanol: 100% isopropanol).
    6. Rinse the slides twice for 2 minutes in a 95% solution of the 100% alcohols.
    7. Place slides in an 80% solution of the 100% alcohols for 2 minutes, followed by deionized water for 5 minutes.
    8. Rinse slides several times with fresh deionized water followed by another five minutes wash using fresh water.


    Sodium Citrate Antigen Retrieval:

    1. Place slides in a glass slide holder and fill in the rest of the rack with blank slides (10 totals) to ensure even heating.
    2. Place rack in 600 ml of 10 mM sodium citrate, pH 6.0 in a glass 2 L-beaker. Mark a line at the top of the liquid on the beaker.
    3. Microwave for 20 min total, replacing evaporated water every 5 min.
    4. Cool slides for 20 min.
    5. Wash 4 X 3 min in ddH2O, and 3 min in 1X PBS.


    Blocking


    1. Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 minutes at room temperature. Wash 3 X in PBS.
    2. Immerse slides in a dish containing blocking buffer (serum from host species of secondary antibody to be used, diluted 1:10 in TBS). Incubate at 37oC for one hour.


    Incubation with Primary Antibodies


    1. Cover the tissue sections with primary antibody diluted in blocking buffer. Antibody is diluted 1:50 and 1:100. Incubate for 1 hour at 37oC.
    2. Blot excess liquid from slides and rinse three times in PBS for five minutes each wash.
    Incubation with Secondary Antibodies
    3. Cover the tissue sections with secondary antibody diluted in blocking buffer according to manufacturer’s instructions. We routinely use prediluted universal secondary antibody (Jackson ImmunoResearch Laboratories). Incubate at 37oC for 30 min.
    4. Blot excess liquid and rinse twice in TBS for five minutes each wash.


    Counterstaining and Visualization

    1. Counterstain with Hematoxylin.
    2. Rinse several times in deionized water. Blot excess water around tissue, then apply one drop of mounting media to tissue and place coverslip over slide. Seal with nail polish.

    Citrate Solutions:
    Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, thereby unmasking the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections and enhancing the staining intensity of antibodies.


    Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):

    Tri-sodium citrate (dihydrate) 2.94 g
    Distilled water 1000 ml
    Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4¢XC for longer storage.
    Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0):
    Citric acid (anhydrous) 1.92 g
    Distilled water 1000 ml
    Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4oC for longer storage.

    Washing Buffer:

    1 X PBS:
    NaCl 8 g
    KCl 0.2 g
    Na2HPO 41.44 g
    KH2PO4 0.24 g
    Distilled Water 800 mL
    Adjust pH to 7.2 with HCl.
    Adjust volume to 1 L with additional H2O


    Normal Serum Blocking Buffer:
    2% serum from host species of secondary antibody (blocking)
    1% BSA (stabilizer)
    0.1% cold fish skin gelatin (blocking)
    0.1% Triton X-100 (penetration enhancer)
    0.05% Tween 20 (detergent and surface tension reducer)
    0.05% sodium azide (preservative)
    Dissolve in 1X PBS
    Mix well and store at 40C.


    Avidin/Biotin Block:
    Avidin 0.001% in 1 X PBS
    Biotin 0.001% in 1 X PBS
    Store these blocking solution at 4oC.


    Primary Antibody Dilution Buffer:
    1%BSA (stabilizer and blocking)
    0.1% cold fish skin gelatin (blocking)
    0.05% sodium azide (preservative)
    0.01M PBS pH7.2

    Note: 1) Antibodies diluted using this buffer can be stored at 4°C for 6 months without reducing binding activity. 2) This buffer cannot be used for diluting HRP conjugated antibodies since sodium azide is an inhibitor of HRP.


    Peroxidase Blocking Solution (3% H2O2 in PBS):
    30% H2O2 2 ml
    1 X PBS - 18 ml
    Mix well and store at 4°C for up to 3 months.
    This solution is recommended for paraffin sections


    References:
    1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov; 41(11):1599-604. PubMed Abstract
    2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61. PubMed Abstract
    3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract
    4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol. 1994 Dec; 174(4):301-7. PubMed Abstract
    5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid. Medicina (B Aires). 54(2):129-32. PubMed Abstract
    6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604. PubMed Abstract
    7. Brown, D., et al. (1996) Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS).Histochem Cell Biol 105:261–267.



    Protocols for ab92378 Anti-HNF4 antibody [EPR3648] and ab129189 Anti-NAPSIN A antibody [EPR6257]:


    1. Solutions and reagents

    1.1 Xylene
    1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%, 50%)
    1.3. Washing buffer/TBST: 1X TBS/0.1% Tween-20, pH to 7.6.
    1.4. Distilled water (dH 2O)
    1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0
    To prepare Antigen Retrieval stock solutions:

    10X Stock: Dissolve 29.4 g sodium citrate trisodium salt dihydrate (C 6H 5Na 3O 7 2H 2O)
    in 1 liter of dH2O. Add 5mL Tween-20.

    1X Working Solution: Mix 200mL 10X stock with 1800mL dH2O; pH to 6.0

    1.6. 3% Hydrogen Peroxide
    1.7. Blocking Buffer: 10% serum in PBS (serum origin depends on the host of the secondary antibody)
    1.8. Primary Antibody Diluent: 5% serum in PBS (serum origin depends on the host of the secondary antibody)
    1.9. Hematoxylin
    1.10. Permanent Mounting Medium



    2. IHC Protocol

    2.1. Deparaffinization/Rehydration
    2.1.1. Heat slides in an oven at 65 °C for 1 hour.
    2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (3 min each), followed by two 100% ethanol rinses (3 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by TBST wash for 3 min on a shaker.


    2.2. Antigen Retrieval

    This is recommended Heat Induced Epitope Retrieval (HIER) using Decloaking Chamber/Pressure Cooker. Hot water bath or Microwave with temperature sensor can be also used (protocol would vary depending on the method used).

    2.2.1. Add 500 ml of dH 2O to Decloaker/Pressure Cooker.
    2.2.2. Immerse slides into staining dish containing Antigen Retrieval Solution. Place staining dish into decloaking chamber.
    2.2.3. Program to run for 30 seconds at 125° C, followed by 10 seconds at 90° C.
    2.2.4. Let it cool down to room temperature (10 - 20 minutes).
    2.2.5. Removes slides and rinse in TBST.
    2.2.6. Proceed to Staining step.


    2.3. Staining

    2.3.1. Wash slides with TBST for 3 min on a shaker.
    2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 5 min.
    2.3.3. Wash slides three times with TBST (3 min each on a shaker).
    2.3.4. Block slides with the blocking solution for 1 hour.
    2.3.5. Dilute primary antibody in primary antibody diluent per recommendation on data sheet.
    2.3.6. Apply primary antibody to each section and incubate overnight in the humidified
    chamber (4 °C).
    2.3.7. Wash slides three times with TBST (3 min each on a shaker).
    2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per manufacturer's recommendation; incubate for 30 min at room temperature.
    2.3.9. Wash slides three times with TBST (5 min each on a shaker).
    2.3.10. Add freshly prepared DAB substrate to the sections and incubate until stain develops (generally 1 min).
    2.3.11. Rinse sections with water.
    2.3.12. Counterstain with Hematoxylin (generally 10 seconds).
    2.3.13. Rinse sections with water.
    2.3.14. Dehydrate samples using two washes with 100% Ethanol (3 min each), followed by two rinses with Xylene (3 min each).

    2.3.15. Mount coverslips on slides using permanent mounting medium.

    Read More

    Abcam Scientific Support

    Answered on Sep 27 2012

    Question

    I kindly ask you to inform me about the amount of reagent (Napsin A, Pax8 and HNF4) required for one analysis (or how many analysis can be done with 1 package of the mentioned reagents).

    Thank you in advance.

    Best regards,

    Read More

    Abcam community

    Verified customer

    Asked on Sep 25 2012

    Answer

    Thank you for your message which has been forwarded to the scientific support team.

    I am sorry it is difficult to let you know the amount of antibody required for one analysis. This will be very individual and will depend on:

    1. The dilution of antibody used, which will need to be optimized by the end user.

    2. The amount of antibody solution added to each sample.

    3. The application being used.

    I can suggest to use the following information from the online datasheets as a guide. This provides the amount of antibody provided in each vial and also the recommended dilutions, which will require optimization for individual experiments:

    ab129189 NAPSIN A 100 µl
    Recommended dilutions:
    WB: 1/1000 - 1/10000.
    IHC-P: 1/100 - 1/250.


    ab92378 HNF4 100 µl
    Reommended dilutions:
    WB: 1/1000 - 1/10000
    IP: 1/10 - 1/100.
    IHC-P: 1/100 - 1/250
    ICC: 1/100 - 1/250.
    Flow Cyt: 1/100.

    ab135618 PAX8 250 ul
    Recomended dilutions.
    WB: 1/100 - 1/500.
    IHC-P: 1/50 - 1/100.
    Flow Cyt: 1/10 - 1/50.

    I hope this will be helpful to you. If you require any further assistance, please let me know which application you are using, and how much antibody solution you will use for each application. If you have any further questions, please do not hesitate to let me know.

    Read More

    Abcam Scientific Support

    Answered on Sep 25 2012

    Question

    Hello, I currently need to know about the lifespans of antibodies, when stored at +4°C and when stored at -20°C. I'm particularly targetting the ab13847, ab22839, ab36055, ab40874 and ab92378 products. Regards,  

    Read More

    Abcam community

    Verified customer

    Asked on Sep 21 2011

    Answer

    Thank you for contacting us. Antibodies are viable for 1 year at 4C and 10 years at -20C. Please check the datasheet of individual antibody for storage instructions. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Abcam Scientific Support

    Answered on Sep 21 2011

    Question

    Which isoforms does this antibody react with? I'm interested in detecting isoforms 1 and 7.

    Read More

    Abcam community

    Verified customer

    Asked on Feb 04 2011

    Answer

    A BLAST search shows that ab92378 can detect isoforms 1 and 7. This antibody can also detect isoforms 2, 4, and 8. Sequence alignment shows that its immunogen shares 100% identity with isoforms 3 and 9. Overall, it is predicted to react with all isoforms. I hope this is helpful. Please contact us again if you need any further information.

    Read More

    Abcam Scientific Support

    Answered on Feb 04 2011

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

    Get resources and offers direct to your inbox Sign up
    A-Z by research area
    • Cancer
    • Cardiovascular
    • Cell biology
    • Developmental biology
    • Epigenetics & Nuclear signaling
    • Immunology
    • Metabolism
    • Microbiology
    • Neuroscience
    • Signal transduction
    • Stem cells
    A-Z by product type
    • Primary antibodies
    • Secondary antibodies
    • Biochemicals
    • Isotype controls
    • Flow cytometry multi-color selector
    • Kits
    • Loading controls
    • Lysates
    • Peptides
    • Proteins
    • Slides
    • Tags and cell markers
    • Tools & Reagents
    Help & support
    • Support
    • Make an Inquiry
    • Protocols & troubleshooting
    • Placing an order
    • RabMAb products
    • Biochemical product FAQs
    • Training
    • Browse by Target
    Company
    • Corporate site
    • Investor relations
    • Company news
    • Careers
    • About us
    • Blog
    Events
    • Tradeshows
    • Conferences
    International websites
    • abcam.cn
    • abcam.co.jp

    Join with us

    • LinkedIn
    • facebook
    • Twitter
    • YouTube
    • Terms of sale
    • Website terms of use
    • Cookie policy
    • Privacy policy
    • Legal
    • Modern slavery statement
    © 1998-2023 Abcam plc. All rights reserved.