Overview

  • Product name
    Anti-PML Protein antibody - N-terminal
    See all PML Protein primary antibodies
  • Description
    Rabbit polyclonal to PML Protein - N-terminal
  • Host species
    Rabbit
  • Specificity
    This antibody detects endogenous levels of total PML protein.
  • Tested applications
    Suitable for: WB, ELISA, IHC-P, ICC/IF, ChIP, IPmore details
  • Species reactivity
    Reacts with: Human, African green monkey
  • Immunogen

    Synthetic peptide corresponding to Human PML Protein aa 39-53 (N terminal).
    Sequence:

    PSPTERAPASEEEFQ


    Database link: P29590

  • Positive control
    • WB: A549 cell extracts. ICC/IF: HeLa cells. ChIP:Chromatin was prepared from modified murine D3 ES cells (an in vitro model of RB1 imprinted expression was generated by integrating human PPP1R26P1 into intron 2 of mouse Rb1 in murine embryonic stem cells (mESCs)). IHC-P: Human normal colon tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab53773 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 1/500 - 1/1000. Detects a band of approximately 100 kDa (predicted molecular weight: 69 kDa).
ELISA 1/10000.
IHC-P Use a concentration of 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF 1/100 - 1/250.
ChIP Use at an assay dependent concentration. PubMed: 24019952
IP Use at an assay dependent concentration. PubMed: 23222847

Target

  • Function
    Key component of PML nuclear bodies that regulate a large number of cellular processes by facilitating post-translational modification of target proteins, promoting protein-protein contacts, or by sequestering proteins. Functions as tumor suppressor. Required for normal, caspase-dependent apoptosis in response to DNA damage, FAS, TNF, or interferons. Plays a role in transcription regulation, DNA damage response, DNA repair and chromatin organization. Plays a role in processes regulated by retinoic acid, regulation of cell division, terminal differentiation of myeloid precursor cells and differentiation of neural progenitor cells. Required for normal immunity to microbial infections. Plays a role in antiviral response. In the cytoplasm, plays a role in TGFB1-dependent processes. Regulates p53/TP53 levels by inhibiting its ubiquitination and proteasomal degradation. Regulates activation of p53/TP53 via phosphorylation at 'Ser-20'. Sequesters MDM2 in the nucleolus after DNA damage, and thereby inhibits ubiquitination and degradation of p53/TP53. Regulates translation of HIF1A by sequestering MTOR, and thereby plays a role in neoangiogenesis and tumor vascularization. Regulates RB1 phosphorylation and activity. Required for normal development of the brain cortex during embryogenesis. Can sequester herpes virus and varicella virus proteins inside PML bodies, and thereby inhibit the formation of infectious viral particles. Regulates phosphorylation of ITPR3 and plays a role in the regulation of calcium homeostasis at the endoplasmic reticulum (By similarity). Regulates transcription activity of ELF4. Inhibits specifically the activity of the tetrameric form of PKM2. Together with SATB1, involved in local chromatin-loop remodeling and gene expression regulation at the MHC-I locus. Regulates PTEN compartmentalization through the inhibition of USP7-mediated deubiquitinylation.
  • Involvement in disease
    Note=A chromosomal aberration involving PML may be a cause of acute promyelocytic leukemia (APL). Translocation t(15;17)(q21;q21) with RARA. The PML breakpoints (type A and type B) lie on either side of an alternatively spliced exon.
  • Sequence similarities
    Contains 2 B box-type zinc fingers.
    Contains 1 RING-type zinc finger.
  • Domain
    Interacts with PKM2 via its coiled-coil domain.
    Binds arsenic via the RING-type zinc finger.
  • Post-translational
    modifications
    Ubiquitinated; mediated by RNF4, SIAH1 or SIAH2 and leading to subsequent proteasomal degradation. 'Lys-6'-, 'Lys-11'-, 'Lys-48'- and 'Lys-63'-linked polyubiquitination by RNF4 is polysumoylation-dependent.
    Undergoes 'Lys-11'-linked sumoylation. Sumoylation on all three sites is required for nuclear body formation. Sumoylation on Lys-160 is a prerequisite for sumoylation on Lys-65. The PML-RARA fusion protein requires the coiled-coil domain for sumoylation. Desumoylated by SENP2 and SENP6. Arsenic induces PML and PML-RARA oncogenic fusion proteins polysumoylation and their subsequent RNF4-dependent ubiquitination and proteasomal degradation, and is used as treatment in acute promyelocytic leukemia (APL).
    Phosphorylated in response to DNA damage, probably by ATR.
    Acetylation may promote sumoylation and enhance induction of apoptosis.
  • Cellular localization
    Nucleus > nucleoplasm. Cytoplasm. Nucleus > PML body. Nucleus > nucleolus. Endoplasmic reticulum membrane. Early endosome membrane. Sumoylated forms localize to the PML nuclear bodies. The B1 box and the RING finger are also required for this nuclear localization. Isoforms lacking a nuclear localization signal are cytoplasmic. Detected in the nucleolus after DNA damage. Sequestered in the cytoplasm by interaction with rabies virus phosphoprotein.
  • Information by UniProt
  • Database links
  • Alternative names
    • Acure promyelocytic leukemia, inducer of antibody
    • MYL antibody
    • Pml antibody
    • PML_HUMAN antibody
    • PP8675 antibody
    • Probable transcription factor PML antibody
    • Promyelocytic leukemia antibody
    • Promyelocytic leukemia inducer of antibody
    • Promyelocytic leukemia protein antibody
    • Protein PML antibody
    • RING finger protein 71 antibody
    • RNF 71 antibody
    • RNF71 antibody
    • TRIM 19 antibody
    • Tripartite motif protein TRIM19 antibody
    • Tripartite motif-containing protein 19 antibody
    see all

Images

  • ab53773 (1/200) staining PML protein in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells (green). Cells were fixed with methanol, permeabilized with triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). Please refer to abreview for further experimental details.

    See Abreview

  • All lanes : Anti-PML Protein antibody - N-terminal (ab53773) at 1/500 dilution

    Lane 1 : A549 (Human lung carcinoma cell line) cell extracts
    Lane 2 : A549 (Human lung carcinoma cell line) cell extracts with immunizing peptide

    Predicted band size: 69 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?

  • Chromatin was prepared from modified murine D3 ES cells (an in vitro model of RB1 imprinted expression was generated by integrating human PPP1R26P1 into intron 2 of mouse Rb1 in murine embryonic stem cells (mESCs)). Cells were fixed with formaldehyde for 10 minutes. Chromatin was sheared into fragment sizes ranging from 200 to 600 bp. For immunoprecipitation 50 μl of sheared chromatin was set aside as input and 20-40 μg of sheared chromatin in a volume of 100 μl was used for antibody incubation. 4 µg of ab53773 to PML was used as a non-related control (black bars) and 4 µl of a rabbit polyclonal to RNAPII CTD repeat YSPTSPS (phospho S5) (shaded bars) was added as the test. The immunoprecipitated DNA was quantified at 3 positions on the the retinoblastoma (Rb1) promoter. 1,2 = Rb1_48; 3,4 = Rb1_69 and 5,6 = Rb1_74

  • ab53773 staining PML in human normal colon tissue. Staining is localized to the nucleus.
    Left panel: ab53773 at 4 µg/ml. Right panel: Isotype control.
    Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved with the EDTA pH 9.0 buffer. Slides were blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with hematoxylin and coverslipped. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

References

This product has been referenced in:
  • Diplas BH  et al. The genomic landscape of TERT promoter wildtype-IDH wildtype glioblastoma. Nat Commun 9:2087 (2018). Read more (PubMed: 29802247) »
  • Zink LM  et al. H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements. Nucleic Acids Res 45:5691-5706 (2017). Read more (PubMed: 28334823) »
See all 22 Publications for this product

Customer reviews and Q&As

11-19 of 19 Abreviews or Q&A

Question

LOT NUMBER GR16227-10
DESCRIPTION OF THE PROBLEM Non-specific staining Data shows too much significant signal even in cytoplasm. It has been known that PML is nuclear localized protein. It seems like background effect.
SAMPLE mouse Sample preparation : cells on coverslip
PRIMARY ANTIBODY Concentration or dilution 1:200 Diluent buffer 5% horse serum Incubation time : 4℃ overnight (16hrs) Wash Buffer : 1XPBS Number of washes 3times for 5mins
DETECTION METHOD Confocal710 (ZEISS LSM series)
POSITIVE AND NEGATIVE CONTROLS USED Positive control : non Negative control : non
ANTIBODY STORAGE CONDITIONS -20℃
FIXATION OF SAMPLE Yes/No : Yes If yes: Fixative agent and concentration : PFA 4% Fixation time : 20mins Fixation temperature : Room temperature
ANTIGEN RETRIEVAL Antigen retrieval method : no
PERMEABILIZATION STEP Permeabilization method: with weak detergent Permeabilizing agent and concentration: added 0.5% triton X-100 in PBS
BLOCKING CONDITIONS Blocking agent (eg BSA, serum…): horse serum in PBS Concentration : 5% Blocking time : 1hr Blocking temperature : Room temperature Endogenous peroxidases blocked? no Endogenous biotins blocked? no
SECONDARY ANTIBODY Alex Fluor donkey anti-rabbit-555 (A31572) Invitrogen Species: donkey Isotype: IgG Reacts against: rabbit Concentration or dilution 1:200 Diluent buffer : 5% horse serum Incubation time : 1hr at RT Fluorochrome or enzyme conjugate : alexa 555 Wash Buffer : 1XPBS Number of washes 3times for 5mins
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? -
ADDITIONAL NOTES Test for co-localization between PML protein and my target protein in Raw264.7 cells (mouse macrophage) with ICC. I’ve gotten optimized data every time using this protocol what I write above. I don’t think the procedure has some problems. It’s a verified protocol. (Ref. Marnefa et al. 2011. MBoC) NA-related nuclear functions of human Pat1b, the P-body mRNA decay factor) I just wonder why I see some spots even in cytoplasm and I can’t see significant spots called PML body published in previous reports and the review in Abcam webpage. International Immunology, Vol. 23, No. 4, pp. 287–296 doi:10.1093/intimm/dxr004 Advance Access publication 22 March 2011 Molecular Biology of the Celllocalization http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E11-05-0415) on November 16, 2011

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. I would like to reassure you that this antibody is tested and covered by our guarantee forICC-IF and mouse samples.

Before deciding how to proceed with this particular case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some details of the procedure.

1. Could you confirm ifthe current vialofsecondary antibody is working well with other antibodies? It would be important to include a no primary control to assess if this is giving any background.

The speckled non specific fluorescence could be from aggregates of the conjugate. I can recommend to try spinning the vial of secondary verybriefly before using (to spin out aggregates).

The concentration of the secondarymay also need to be reduced in order to help optimize the results.

2. Dust on slides, or particles in the buffers can autofluorescence and can give a speckled staining appearance. I can suggest to ensure the slides and buffers are as clean as possible. Also, this could be caused by cell debris, and so I can recommend to ensure the cell culture sample is as healthy as possible.

3. I can recommend to fix for 10 minutes only to reduce the amount of protein crosslinking, which could affect staining.

4. For the wash steps, try washing in buffer containing 0.2% Tween 20. This will help to wash away any excess antibody. Also include 0.2% Tween 20 in the antibody dilution buffer.

In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested information and details of how you would like to proceed.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (U2OS human osteosarcoma cell line)
Specification
U2OS human osteosarcoma cell line
Fixative
Formaldehyde
Permeabilization
Yes - triton 0,5% in PBS for 5 min
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jan 20 2012

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (WI38 human fibroblasts)
Specification
WI38 human fibroblasts
Fixative
Formaldehyde
Permeabilization
Yes - triton 0,5% 5min
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jan 19 2012

Answer

Thank you for contacting us. The PML antibody ab53773 is raised against a peptide derived from the 20 amino acid range from aa35 to aa55 of human PML, Uniprot P29590 (PML_HUMAN). The exact sequence of the peptide is considered proprietary. http://www.Uniprot.org/uniprot/P29590 This peptide sequence is present in all isoforms of human PML listed in the Uniprot entry. Translocation to the nucleus is believed to be dependent on sumoylation of PML by SUMO-1, as discussed in the reference below. Assuming none of these modifications are to the immunogen sequence itself, antibody ab53773 should be capable of recognizing all isoforms, with and without these modifications, and so it should be capable of recognizing both nuclear and cytoplasmic PML. However, this has not to our knowledge been confirmed experimentally. J Cell Sci. 1999 Feb;112 ( Pt 3):381-93. SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation. PMID: 9885291 http://jcs.biologists.org/content/112/3/381.full.pdf 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
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Jurkat)
Specification
Jurkat
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.2% Triton X-100 in PBS

Mr. Benny Garfinkel

Verified customer

Submitted Dec 29 2010

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Fibroblasts)
Specification
Fibroblasts
Fixative
Paraformaldehyde
Permeabilization
Yes - Methanol, 30 min at -20°C
Blocking step
FCS as blocking agent for 30 minute(s) · Concentration: 20% · Temperature: RT°C

Dr. Ioannis Gavvovidis

Verified customer

Submitted Dec 10 2010

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HaCaT keratinocyte)
Specification
HaCaT keratinocyte
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.25% triton X-100
Blocking step
2.5% BSA plus 1% goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jun 05 2009

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Embryonic Skin, epidermal keratinocyets,)
Specification
Embryonic Skin, epidermal keratinocyets,
Fixative
Formaldehyde
Permeabilization
Yes - 1%TritonX-100/0.2%Saponin

Dr. Andrei Mardaryev

Verified customer

Submitted Aug 15 2008

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Methanol
Permeabilization
Yes - 0.5% TritonX100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Nov 09 2007

11-19 of 19 Abreviews or Q&A

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