Overview

  • Product name
  • Description
    Rabbit polyclonal to PHF8
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-P, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 800 - 900 of Human PHF8.

    Read Abcam's proprietary immunogen policy (Peptide available as ab36067.)

  • Positive control
    • This antibody gave a positive signal in the following whole cell lysates: HeLa; U2OS; Jurkat; HepG2.

Properties

Applications

Our Abpromise guarantee covers the use of ab36068 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
ICC/IF
IHC-P
ChIP
  • Application notes
    ICC/IF: Use at a concentration of 5 µg/ml.
    IHC-P: Use at a concentration of 10 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
    WB: Use at a concentration of 1 µg/ml. Detects a band of approximately 125 & 140 kDa (predicted molecular weight: 118 kDa).


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    • Function
      Histone lysine demethylase with selectivity for the di-and monomethyl states that plays a key role cell cycle progression, rDNA transcription and brain development. Demethylates mono- and dimethylated histone H3 'Lys-9' residue (H3K9Me1 and H3K9Me2), dimethylated H3 'Lys-27' (H3K27Me2) and monomethylated histone H4 'Lys-20' residue (H4K20Me1). Acts as a transcription activator as H3K9Me1, H3K9Me2, H3K27Me2 and H4K20Me1 are epigenetic repressive marks. Involved in cell cycle progression by being required to control G1-S transition. Acts as a coactivator of rDNA transcription, by activating polymerase I (pol I) mediated transcription of rRNA genes. Required for brain development, probably by regulating expression of neuron-specific genes. Only has activity toward H4K20Me1 when nucleosome is used as a substrate and when not histone octamer is used as substrate. May also have weak activity toward dimethylated H3 'Lys-36' (H3K36Me2), however, the relevance of this result remains unsure in vivo. Specifically binds trimethylated 'Lys-4' of histone H3 (H3K4me3), affecting histone demethylase specificity: has weak activity toward H3K9Me2 in absence of H3K4me3, while it has high activity toward H3K9me2 when binding H3K4me3.
    • Involvement in disease
      Defects in PHF8 are the cause of mental retardation syndromic X-linked Siderius type (MRXSSD) [MIM:300263]. A disorder characterized by mild to borderline mental retardation with or without cleft lip/cleft palate.
    • Sequence similarities
      Belongs to the JHDM1 histone demethylase family. JHDM1D subfamily.
      Contains 1 JmjC domain.
      Contains 1 PHD-type zinc finger.
    • Domain
      The PHD-type zinc finger mediates the binding to H3K4me3. Binding to H3K4me3 promotes its access to H3K9me2.
      The linker region is a critical determinant of demethylase specificity. It enables the active site of JmjC to reach the target H3K9me2 when the PHD-type zinc finger binds to H3K4me3.
    • Post-translational
      modifications
      Phosphorylation at Ser-69 and Ser-120 are required for dissociation from chromatin and accumulation of H4K20Me1 levels during prophase.
    • Cellular localization
      Nucleus. Nucleus > nucleolus. Recruited to H3K4me3 sites on chromatin during interphase. Dissociates from chromatin when cells enter mitosis.
    • Information by UniProt
    • Database links
    • Alternative names
      • Histone lysine demethylase PHF8 antibody
      • JHDM1F antibody
      • Jumonji C domain containing histone demethylase 1F antibody
      • MRXSSD antibody
      • PHD finger protein 8 antibody
      • PHF8 antibody
      • PHF8_HUMAN antibody
      • ZNF422 antibody
      see all

    Images

    • Chromatin was prepared from SupT1 (human T-cell leukemia cell line) nuclear lysate. Cross-linking (X-ChIP) with 1% formaldehyde for 10 minutes. ChIP was performed with ab36068 at 0.1 μg/μg chromatin. Positive control: Transcriptional start site (TSS). Negative control: cells treated with PHF8-specific shRNA. The immunoprecipitated DNA was quantified by real time PCR. 

      See Abreview

    • ICC/IF image of ab36068 stained HeLa 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 (ab36068, 5µg/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. This antibody also gave a positive result in 4% formaldehyde fixed (10 min) HepG2 cells at 5µg/ml, and in 100% methanol fixed (5 min) Hek293 cells at 5µg/ml.
    • All lanes : Anti-PHF8 antibody (ab36068) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
      Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
      Lane 4 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 118 kDa
      Observed band size: 140 kDa
      why is the actual band size different from the predicted?


      Exposure time: 2 minutes
    • IHC image of ab36068 staining in human skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab36068, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
    • All lanes : Anti-PHF8 antibody (ab36068) at 1 µg/ml

      Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
      Lane 2 : U2OS (Human osteosarcoma cell line) Whole Cell Lysate
      Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
      Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 118 kDa
      Observed band size: 125,140 kDa why is the actual band size different from the predicted?

    References

    This product has been referenced in:
    • Chen X  et al. Phf8 histone demethylase deficiency causes cognitive impairments through the mTOR pathway. Nat Commun 9:114 (2018). IHC (PFA fixed) ; Mouse . Read more (PubMed: 29317619) »
    • Shao P  et al. Histone demethylase PHF8 promotes epithelial to mesenchymal transition and breast tumorigenesis. Nucleic Acids Res 45:1687-1702 (2017). ChIP ; Human . Read more (PubMed: 27899639) »
    See all 13 Publications for this product

    Customer reviews and Q&As

    1-9 of 9 Abreviews or Q&A

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (293T)
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis Tris)
    Loading amount
    20 µg
    Specification
    293T
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Oct 23 2018

    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Mouse embryonic fibroblasts)
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-tris)
    Loading amount
    20 µg
    Specification
    Mouse embryonic fibroblasts
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Oct 23 2018

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (293T)
    Gel Running Conditions
    Reduced Denaturing (4-20%)
    Loading amount
    50 µg
    Specification
    293T
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Nov 07 2014

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Primary cells, testicular germ cells)
    Permeabilization
    Yes - Triton X-100
    Specification
    Primary cells, testicular germ cells
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 0.5% · Temperature: RT°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Jul 30 2013

    Answer

    I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab84779.

    To check the status of the order please contact our Customer Service team and reference this number.

    Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

    I wish you the best of luck with your research.

    Read More

    Answer

    Thank you for your reply. I am glad to hear your are able to get the correct band.

    Cross linking should not interfere with the IP; however cross-linking is a time-critical procedure. Cross-linking should generally only be carried out for a few minutes. Excessive cross-linking can lead to a decrease in the amount of protein bound to the DNA, reduction in the availability of epitopes/changes in epitopes for antibody binding and, in turn, reductions in the material bound/antigen availability in your sample.It is recommended to always perform a time-course experiment to optimize cross-linking conditions.



    There are a number of advantages and disadvantages to both procedures our ChIP tips page details these as well as answers to a number of common ChIP questions. That page is located athttps://www.abcam.com/index.html?pageconfig=resource&rid=310


    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Answer

    Thank you for contacting us. I am sorry to hear that you have been experiencing issues when in ChIP using this product. According to the Uniprot information for this protein (http://www.uniprot.org/uniprot/Q80TJ7)this product may recognize the ~114 kDa Isoform 1 or the 89 kDa Isoform 2 of PHF8 in mouse. These figures are based on sequence and may not reflect actual size in real world experiments; however this does align with the 111 kDa bands which you have been seeing. While this product has been predicted to work in mouse based on sequence homology, to our knowledge this has not been tested before. I have run a BLAST of the immunogen sequence against mouse with the following result: gi|123285641|emb|CAM17163.1| PHD finger protein 8 [Mus musculus] Length=820 GENE ID: 320595 Phf8 | PHD finger protein 8 [Mus musculus] Score = 46.9 bits (103), Expect = 4e-05, Method: Composition-based stats. Identities = 13/14 (93%), Positives = 13/14 (93%), Gaps = 0/14 (0%) Have you attempted an x-ChIP protocol? We do have a customer review using this antibody in X-ChIP on human cells which you may find under the Abreview tab of the datasheet on the website. X-ChIP may be more suitable when analyzing proteins that have either a weaker DNA affinity or are a long way from DNA. Cross-linking may be required to stop proteins dissociating from the DNA. Histones are tightly associated therefore N-ChIP can be performed when studying histones. It is also possible that there is not enough antibody included in the immunoprecipitation.We would suggest using between 3-5 μg of antibody in the first instance. This could be increased to 10ug if no signal is observed. I have also included our ChIP protocols for your review. We also offer our EpiSeeker One Step ChIP kit which is guaranteed to work in mouse. More information about our EpiSeeker kits and line may be found at these links: https://www.abcam.com/EpiSeeker-ChIP-Kit-One-Step-ab117138.html https://www.abcam.com/index.html?pageconfig=resource&rid=14469 I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Use our products? Submit an Abreview. Earn rewards! https://www.abcam.com/abreviews

    Read More
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    ChIP
    Sample
    Human Cell lysate - nuclear (T-cell leukemia (SupT1))
    Negative control
    PHF8 specific shRNA treated cells
    Specification
    T-cell leukemia (SupT1)
    Detection step
    Real-time PCR
    Type
    Cross-linking (X-ChIP)
    Duration of cross-linking step: 10 minute(s) and 0 second(s)
    Specification of the cross-linking agent: Formaldehyde 1%
    Positive control
    Transcriptional Start site (TSS)

    Abcam user community

    Verified customer

    Submitted Apr 18 2012

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HPB-ALL (T-cell Leukemia), HeLa cells)
    Gel Running Conditions
    Reduced Denaturing (7.5%)
    Loading amount
    20 µg
    Treatment
    PHF8 specific shRNA
    Specification
    HPB-ALL (T-cell Leukemia), HeLa cells
    Blocking step
    Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Apr 10 2012

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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