Overview

  • Product name

    Anti-Perforin antibody [B-D48]
    See all Perforin primary antibodies
  • Description

    Mouse monoclonal [B-D48] to Perforin
  • Host species

    Mouse
  • Tested applications

    Suitable for: WB, Flow Cyt, IHC-Fr, IHC-Pmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant human perforin.

  • Positive control

    • IHC-P: Human spleen FFPE tissue sections.

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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    Constituent: PBS

    Sterile-filtered through 0.22 µm and treated to remove endotoxins.
  • Concentration information loading...
  • Purity

    Ion Exchange Chromatography
  • Clonality

    Monoclonal
  • Clone number

    B-D48
  • Myeloma

    x63-Ag8.653
  • Isotype

    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab47225 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 Use a concentration of 1 µg/ml. Predicted molecular weight: 70 kDa.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

IHC-Fr Use a concentration of 20 µg/ml.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function

    Plays a key role in secretory granule-dependent cell death, and in defense against virus-infected or neoplastic cells. Plays an important role in killing other cells that are recognized as non-self by the immune system, e.g. in transplant rejection or some forms of autoimmune disease. Can insert into the membrane of target cells in its calcium-bound form, oligomerize and form large pores. Promotes cytolysis and apoptosis of target cells by facilitating the uptake of cytotoxic granzymes.
  • Involvement in disease

    Defects in PRF1 are the cause of hemophagocytic lymphohistiocytosis familial type 2 (FHL2) [MIM:603553]; also known as HPLH2. Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous, rare autosomal recessive disorder. It is characterized by immune dysregulation with hypercytokinemia and defective natural killer cell function. The clinical features of the disease include fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia, and neurological abnormalities ranging from irritability and hypotonia to seizures, cranial nerve deficits, and ataxia. Hemophagocytosis is a prominent feature of the disease, and a non-malignant infiltration of macrophages and activated T lymphocytes in lymph nodes, spleen, and other organs is also found.
  • Sequence similarities

    Belongs to the complement C6/C7/C8/C9 family.
    Contains 1 C2 domain.
    Contains 1 EGF-like domain.
    Contains 1 MACPF domain.
  • Domain

    The C2 domain mediates calcium-dependent binding to lipid membranes. A subsequent conformation change leads to membrane insertion of beta-hairpin structures and pore formation. The pore is formed by transmembrane beta-strands.
  • Post-translational
    modifications

    N-glycosylated.
  • Cellular localization

    Cytoplasmic granule lumen. Secreted. Cell membrane. Endosome lumen. Stored in cytoplasmic granules of cytolytic T-lymphocytes and secreted into the cleft between T-lymphocyte and target cell. Inserts into the cell membrane of target cells and forms pores. Membrane insertion and pore formation requires a major conformation change. May be taken up via endocytosis involving clathrin-coated vesicles and accumulate in a first time in large early endosomes.
  • Information by UniProt
  • Database links

  • Alternative names

    • Cytolysin antibody
    • FLH2 antibody
    • HPLH2 antibody
    • Lymphocyte pore-forming protein antibody
    • P1 antibody
    • PERF_HUMAN antibody
    • perforin 1 (pore forming protein) antibody
    • Perforin 1 antibody
    • Perforin-1 antibody
    • PFP antibody
    • PGFL antibody
    • PIGF antibody
    • PIGF-2 antibody
    • PLGF antibody
    • Pore forming protein antibody
    • prf1 antibody
    • SHGC-10760 antibody
    see all

Images

  • IHC image of Perforin staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab47225, 10µg/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.

References

This product has been referenced in:

  • Konjevic Sabolek M  et al. Communication of CD8+ T cells with mononuclear phagocytes in multiple sclerosis. Ann Clin Transl Neurol 6:1151-1164 (2019). Read more (PubMed: 31353869) »
  • Bengsch B  et al. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns. J Immunol Methods 453:3-10 (2018). Read more (PubMed: 28322863) »
See all 9 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

You are welcome.

We do not have any data for cross-reactivity with non-primate samples. I tried finding an amino acid sequence for non-primate perforin to align with human perforin, which was the immunogen for ab47225.

The unreviewed UniProt entry for a species of macaque,

http://www.uniprot.org/uniprot/B3FWV1

is 95% identical with the sequence of human perforin

(http://www.uniprot.org/uniprot/P14222),

which suggests that, at least for this species, cross-reactivity is likely.

Read More

Answer

Thank you for sending the details of your protocol.

I think for PFA-fixed, paraffin-embedded tissue, your protocol is correct. However, our source for this clone has told us that it was only tested in the past for IHC on frozen tissue by collaborators, and they did not provide a protocol. I suspect they may have used a coagulative fixative such as acetone or methanol. These are typically used to preserve tissue before freezing, as they do not introduce chemical modification to proteins, unlike formaldehyde-based fixatives. These modification tend to interfere with antibody:antigen recognition. The retrieval step aims to reverse this, but is not always effective.

I can suggest trying an enzymatic retrieval instead of a heat-mediated retrieval (trypsin or proteinase K), but we have no data indicating this will or will not work. I do suggest obtaining a positive control of spleen tissue, to confirm that the results are negative due to the procedure or antibody, and not due to lack of expression.

Read More

Answer

Thank you for bringing this to our attention. To our knowledge, no antigen retrieval should be necessary for histochemistry on frozen tissue sections, or permeabilization assuming your sections are no thicker than 10 microns.

Can you please tell me what your tissue samples are (tissue type and species), how it is fixed, and how much antibody you are incubating with the sections and for how long? Are you confident the secondary antibody is effective with mouse IgG? If you have an image of the staining, could you please send it?

I look forward to your reply.

Read More

Answer

Thank you for contacting us. I have doublechecked with the lab and they have confirmed that the antibody ab47225 is provided just in PBS, without any carrier protein or preservative. Format: Phosphate-buffered saline. Sterile-filtered through 0.22 µm. Carrier and preservative free. 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

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