Recombinant
RabMAb

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

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, Flow Cyt, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human HNF-4-alpha aa 1-100. The exact sequence is proprietary.

  • Positive control
    • WB: HepG2, A549 and SW480 cell lysates. IHC-P: Human colon and kidney tissues. ICC/IF: HepG2 cells. Flow Cyt: HepG2 cells.
  • General notes

    A trial size is available to purchase for this antibody.

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

    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.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

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 1/2000. Predicted molecular weight: 53 kDa.

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

IHC-P 1/250 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

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

Flow Cyt 1/70.

For unpurified use at 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/100 - 1/250.
  • 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.
    • Information by UniProt
    • Database links
    • 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

    • 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) 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 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).

    • 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.

    • 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) 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.

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

    • 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.

    • Overlay histogram showing HepG2 cells stained with unpurified ab92378 (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 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. 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.

    References

    This product has been referenced in:
    • Jiang Y  et al. H19 Is Expressed in Hybrid Hepatocyte Nuclear Factor 4a+ Periportal Hepatocytes but Not Cytokeratin 19+ Cholangiocytes in Cholestatic Livers. Hepatol Commun 2:1356-1368 (2018). Read more (PubMed: 30411082) »
    • Skrzypczyk A  et al. Noncoding RNA Transcripts during Differentiation of Induced Pluripotent Stem Cells into Hepatocytes. Stem Cells Int 2018:5692840 (2018). Read more (PubMed: 30210551) »
    See all 5 Publications for this product

    Customer reviews and Q&As

    1-7 of 7 Abreviews or Q&A

    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

    Mr. David Ivancic

    Verified customer

    Submitted May 21 2018

    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

    Abcam user community

    Verified customer

    Submitted Aug 09 2016

    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

    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

    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

    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

    Question
    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

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

    Sign up