Overview

  • Product name
  • Description
    Rabbit polyclonal to HIPK2
  • Host species
    Rabbit
  • Specificity
    ab28507 recognises HIPK2. It may recognize isoform 1, 2 and 3 of HIPK2 as the immunogen sequence is present in all three isoforms. Please refer to the literature for more information on the three isforms. It does not recognize HIPK1, HIPK3, or HIPK4.
  • Tested applications
    Suitable for: ICC/IF, ELISA, WB, IHC-P, IHC-Fr, ChIPmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat, Cow, Dog, Zebrafish
  • Immunogen

    Synthetic peptide derived from within residues 37 - 86 of human HIPK2.

    Read Abcam's proprietary immunogen policy .

  • Positive control
    • HepG2 Cell Lysate, human heart tissue

Properties

Applications

Our Abpromise guarantee covers the use of ab28507 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
ICC/IF Use a concentration of 1 µg/ml.
ELISA 1/62500.
WB Use a concentration of 0.06 µg/ml. Predicted molecular weight: 110 kDa.Can be blocked with Human HIPK2 peptide (ab111675). Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.
IHC-P Use a concentration of 4 - 8 µg/ml.
IHC-Fr Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration. PubMed: 23565059

Target

  • Function
    Protein kinase acting as a corepressor of several transcription factors, including SMAD1 and POU4F1/Brn3a and probably NK homeodomain transcription factors. Inhibits cell growth and promotes apoptosis. Involved in transcriptional activation of TP53 and TP73. Phosphorylation of TP53 may be mediated by a TP53-HIPK2-AXIN1 complex. In response to TGFB, cooperates with DAXX to activate JNK. Phosphorylates the antiapoptotic factor CTBP1 and promotes its proteasomal degradation. In the Wnt/beta-catenin signaling pathway acts as an intermediate kinase between TAK1 and NLK to promote the proteasomal degradation of MYB (By similarity). Phosphorylates CBX4 upon DNA damage and promotes its E3 SUMO-protein ligase activity. PML, HIPK2 and FBXO3 may act synergically to activate p53/TP53-dependent transactivation.
  • Tissue specificity
    Highly expressed in heart, muscle and kidney. Weakly expressed in a ubiquitous way. Down-regulated in several thyroid and breast tumors.
  • Sequence similarities
    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. HIPK subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Phosphorylated on tyrosines (By similarity). Autophosphorylated.
    Sumoylated. When conjugated it is directed to nuclear speckles. Desumoylated by SENP1 (By similarity). Sumoylation on Lys-32 is promoted by the E3 SUMO-protein ligase CBX4.
    Ubiquitinated by FBXO3, leading to rapid proteasome-dependent degradation. This degradation, but not ubiquitination, is prevented in the presence of PML.
  • Cellular localization
    Nucleus > PML body. Cytoplasm. Concentrated in PML/POD/ND10 nuclear bodies. Small amounts are cytoplasmic.
  • Information by UniProt
  • Database links
  • Alternative names
    • hHIPk 2 antibody
    • hHIPk2 antibody
    • HIPK 2 antibody
    • Hipk2 antibody
    • HIPK2_HUMAN antibody
    • Homeodomain interacting protein kinase 2 antibody
    • Homeodomain-interacting protein kinase 2 antibody
    • Nbak1 antibody
    • Nuclear body-associated kinase 1 antibody
    • PRO0593 antibody
    • Sialophorin tail-associated nuclear serine/threonine-protein kinase antibody
    • Stank antibody
    see all

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pineal tissue labelling HIPK2 with ab28507 at 1/100. A Cy3-conjugated donkey anti-rabbit (1/200) was used as the secondary antibody. Positive staining seen the nuclei of pinealocytes and intersticial cells. Magnification: 20X. Exposure time: 0.5 - 2.0 seconds. Left - DAPI. Middle - HIPK2. Right - Merge.
  • ab28507, at 4.0-8.0ug/ml, staining human HIPK2 in (Human heart, cells with positive label are Myocardial cells) by Immunohistochemistry, Paraffin embedded tissue.
  • All lanes : Anti-HIPK2 antibody (ab28507) at 1/10000 dilution

    Lane 1 : Control - HeLa Whole Cell extract
    Lane 2 : HIPK2 plasmid overexpression

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : AlexaFluor 680 goat anti-rabbit at 1/10000 dilution

    Predicted band size: 110 kDa



    Samples were incubated in primary antibody for 14 hours at 4°C.

     

    See Abreview

  • Anti-HIPK2 antibody (ab28507) at 0.06 µg/ml + HepG2 Cell Lysate

    Secondary
    Rabbit Anti HIPK2 at 1/50000 dilution

    Predicted band size: 110 kDa

  • ICC/IF image of ab28507 stained MCF7 cells. 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 (ab28507, 1µg/ml) 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.

References

This product has been referenced in:
  • Oh HJ  et al. Inhibition of the processing of miR-25 by HIPK2-Phosphorylated-MeCP2 induces NOX4 in early diabetic nephropathy. Sci Rep 6:38789 (2016). Read more (PubMed: 27941951) »
  • Singh SP  et al. HIF-1a Plays a Critical Role in the Gestational Sidestream Smoke-Induced Bronchopulmonary Dysplasia in Mice. PLoS One 10:e0137757 (2015). IHC-P, WB ; Mouse . Read more (PubMed: 26361040) »
See all 5 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Question
Answer

I am very pleased to hear you would like to accept our offer and test ab28507 in IHC-P on mouse. This code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for IHC-P on mouse and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa cells)
Loading amount
30 µg
Specification
HeLa cells
Gel Running Conditions
Reduced Denaturing (10% Tris-Gly gel)
Blocking step
LI-COR® Odyssey® Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Dec 21 2011

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (mouse retina sections)
Specification
mouse retina sections
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Nov 22 2010

Answer

Thank you for your reply. The originator of the antibody assures me that the antibody does not react with HIPK1, HIPK3, or HIPK4. I include below the exact protocol that was used to test the antibody in Western blot: To prepare total cell lysates, spin down cells and re-suspension the pellet in PBS to make the final concentration around 3 x 106cells/ ml. This is a convenient cell density for many cell lines, but adjustments may be necessary for cell types that differ substantially in size and protein content. Prepare cell extracts in appropriate non-reducing or reducing sample buffer as indicated on the experiment or product date sheet. In some cases reducing agents may disrupt the conformation that is recognized by a monoclonal detection antibody. Mix the cell suspension with an equal volume of non-reducing 2X SDS gel sample buffer (6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, and bromophenyl blue) or reducing 2X SDS gel sample buffer [non-reducing buffer plus 20 mM dithithreitol (DTT)]. Sonicate the cells to fragment the DNA using 8-10 bursts of 2-3 seconds each. 1. Load cell extracts and separate proteins on an appropriate percentage SDS polyacrylamide gel (SDS-PAGE) (smaller proteins should be run on a higher percentage gel and higher molecular weight proteins should be run on a lower percentage gel). 2. Transfer the separated proteins onto a membrane and incubate the membrane for 1 hour at room temperature or overnight at 2-8 °C in Blocking Solution (1 X PBS, pH 7.4 containing 5% dry milk). 3. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer (1X PBS with 0.1% NP40,). 4. Incubate the membrane for 2 hours at room temperature or overnight at 2-8 °C in Blotting Buffer (1 X PBS, pH 7.4 containing 5% dry milk unless otherwise indicated) containing primary antibody ( concentration as indicated on datasheet). 5. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer. 6. Incubate the membrane at room temperature for 1 hour in Blotting Buffer containing an appropriate secondary reagent such as goat anti rabbit IgG or anti-mouse IgG -HRP at 1:10,000. 7. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer. 8. Detect with chemiluminescence reagents. 9. Optimal dilutions should be determined by each laboratory for each application. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. ab28507 recognizes HIPK2 and was tested on HepG2 cells. It may recognize isoforms 1, 2 and 3 as the immunogen sequence is present in all three isoforms. Isoform two is missing approximately 300 amino acids and would run close to 80 kDa. Please refer to the literature for more information on the three isoforms. It is not clear to me how these three isoforms are regulated. It is conceivable that the HepG2 cells could express more or less of a different isoform depending on how they are cultured or maintained. I am also not certain how the expression of the different isoforms varies in regards to liver (HepG2) versus kidney (293) cells. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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