Anti-Perforin antibody [CB5.4] (ab16074)

Reviews (3)Q&A (5)References (17)

Overview

  • Product name
    Anti-Perforin antibody [CB5.4]
    See all Perforin primary antibodies
  • Description
    Rat monoclonal [CB5.4] to Perforin
  • Host species
    Rat
  • Tested applications
    Suitable for: WB, IHC-Fr, IHC-P, ICC, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse
  • Immunogen

    Recombinant fragment, corresponding to amino acids 98 - 534 of Mouse Perforin.

  • Epitope
    This antibody recognizes mouse perforin, region amino acids 402-534.

Properties

Applications

Our Abpromise guarantee covers the use of ab16074 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. Detects a band of approximately 66 kDa (predicted molecular weight: 66 kDa).
IHC-Fr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
ICC Use a concentration of 5 µg/ml.
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Perforin antibody [CB5.4] (ab16074)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Perforin antibody [CB5.4] (ab16074)This image is courtesy of an anonymous Abreview

      ab16074 staining Perforin in Mouse lymphoid tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with a zinc-based fixative and blocked with rodent block-M for 20 minutes at 20°C. Samples were incubated with primary antibody (1/50 in TBS + 0.05% Tween20) for 1 hour at 20°C. A HRP-conjugated Goat polyclonal was used as the secondary antibody.

      See Abreview

    References

    This product has been referenced in:
    • Wijesundara DK  et al. Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice. J Virol 92:N/A (2018). Read more (PubMed: 29437963) »
    • Kenchegowda D  et al. Inactivation of maternal Hif-1a at mid-pregnancy causes placental defects and deficits in oxygen delivery to the fetal organs under hypoxic stress. Dev Biol 422:171-185 (2017). Read more (PubMed: 27940158) »
    See all 17 Publications for this product

    Publishing research using ab16074? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-8 of 8 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Perforin antibody [CB5.4]

     Good
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (spleen)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Tris/EDTA pH 9.0
    Permeabilization
    No
    Specification
    spleen
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 15% · Temperature: RT°C
    Fixative
    10% NBF
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 27 2018

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Perforin antibody [CB5.4]

     Good
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Sample
    Mouse Cell (uterus, placenta)
    Specification
    uterus, placenta
    Permeabilization
    No
    Fixative
    10% neutral buffered formalin
    Read More

    Dr. Varkha Agrawal

    Verified customer

    Submitted Jul 03 2013

    Question

    Phone call requesting information on which antigen retrieval would be recommended to use the Anti-Perforin antibody [CB5.4] (ab16074).

    Read More
    Answer

    I can confirm that the Anti-Perforin antibody [CB5.4] (ab16074) has been used with citrate buffer (pH 6) when performing staining of paraffin embedded sections. I would however advise that it may be worthwhile to optimize this for your particular tissue preparations. Therefore using both citrate buffer (pH 6) as well as Tris-EDTA (pH 9) and seeing which provides the best results for you.

    Read More

    Question

    ein kostenloser Antikörper wäre mir lieber. Wenn es möglich ist, dass ich mir diesen aussuchen kann, dann würde ich mich für folgenden entscheiden: https://www.abcam.com/F4-80-antibody-CI-A3-1-Allophycocyanin-ab105080.html.

    Read More
    Answer

    Vielen Dank für Ihre Anfrage.

    Es tut mir leid, dass Sie Probleme mit diesem Antikörper hatten. Wie besprochen habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer 123456 und solltemorgen bei Ihnen ankommen.

    Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

    Read More

    Question

    Hier das ausgefüllte questionnaire, im Anhang die Bilder (nur der rote Kanal =Perforin):

    2nd rab rat ist das Bild der Kontrollfärbung, wo nur 2. Antikörper gefärbt wurden. Die Zahlen Perf-50 heißt, dass der Primärantikörper gegen Perforin in einer Verdünnung von 1:50 verwendet wurde.

    Das Bild 12-08-07 spleen perf 50 cd8 50 stammt aus der ersten Färbung (Titration 1) die beiden anderen aus der 2. Färbung



    1) Abcam product code ab 16074



    2) Abcam order reference number or product batch number



    3) Description of the problem

    not sure whether the signal is just background, since control without primary antibodies but with secondary antibodies also gives a signal (see attached pictures)



    4) Sample preparation:

    Species mouse, spleen embedded in tissue tek, frozen at -80°C

    Type of sample: Fresh frozen sections (5µm)

    Sample preparation: fixed in acetone for 10 min at RT, encirceled with DAKO pen, blocked with serum, incubation with primary antibodies, washing, incubation with secondary antibodies, washing, incubation with Hoechst, washing, cover slipping with Glycerin/Gelatine

    Positive control staining of spleen

    Negative control only secondary antibodies



    5) Fixation step

    Yes

    If yes: fixed in acetone

    Fixation time10 min

    Fixation temperature RT



    6) Antigen retrieval method

    none



    7) Permeabilization method: no

    Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?

    Permeabilizing agent and concentration:





    8) Blocking agent (eg BSA, serum…): serum (goat or chicken, depending on the second antibody)

    Concentration 1/100

    Blocking time at least 10 min

    Blocking temperature RT



    9) Endogenous peroxidases blocked? no

    Endogenous biotins blocked? no



    10) Primary antibody (If more than one was used, describe in “additional notes”) :

    Concentration or dilution 1:10, 1:20, 1:50, 1:50, 1:500, 1:1000

    Diluent buffer PBS

    Incubation time 1 hour



    11) Secondary antibody: anti-rat Alexa 594 (invitrogen A21471)

    Species:

    Reacts against: rat

    Concentration or dilution 1:200

    Diluent buffer PBS

    Incubation time 1 hour

    Fluorochrome or enzyme conjugate Alexa 594



    12) Washing after primary and secondary antibodies:

    Buffer: PBS

    Number of washes 2 times in a PBS bath



    13) Detection method



    14) How many times have you run this staining? 2 titrations 1.: 1:50, 1:500, 1:1000, 2.: 1:10, 1:20, 1;50

    Do you obtain the same results every time? no

    What steps have you altered to try and optimize the use of this antibody?

    altered washing steps for 2nd titration (did not dip slides into PBS bath but dribbled PBS on top and tapped it off again, also 2 times



    Falls Sie noch etwas benötigen, werde ich es Ihnen zukommen lassen.



    Vielen Dank für Ihre Hilfe,

    Read More
    Answer

    Vielen Dank für Ihre Antwort und für diese weiteren Informationen.

    Da Ihrem Protokoll bis auf Kleinigkeiten nichts hinzuzufügen ist, muss ich leider davon ausgehen, dass der Antikörper nicht so funktioniert hat, wie er sollte.

    Ich weiß, dass Sie viel Zeit für diese Experimente verwendet haben und möchte Ihnen daher einen kostenlosen Antikörperoder einen Gutschein als Ersatz anbieten. Würden Sie mir bitte mitteilen, welche Alternative Sie bevorzugen?

    Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich bald wieder von Ihnen zu hören.

    Read More

    Question

    Concentration is way too high in IHC-Fr (1/10).
    Isotyp controll?

    Read More
    Answer

    Vielen Dank für Ihren Anruf.

    Es tut mir leid zu hören, dass Sie Probleme mit diesem Antikörper haben.

    Ich habe unseren Fragebogen als Word-Dokument an diese E-Mail angehängt. Durch das Ausfüllen des Fragebogens erhalten wir alle nötigen Informationen über Ihre Proben und Ihr Protokoll. Sobald Sie dieses Formular an uns zurückgeschickt haben, werden wir uns Ihr Protokoll ansehen und möglichst Veränderungsvorschläge machen, die Ihre Ergebnisse verbessern werden. Falls sich herausstellt, dass der Antikörper nicht so funktioniert, wie auf dem Datenblatt beschrieben und er innerhalb der letzten 180 Tage gekauft wurde, werden wir Ihnen gerne einen Ersatz oder eine Gutschrift schicken.



    Ich freue mich, bald wieder von Ihnen zu hören.

    Read More

    Question

    ICC/IF with mouse T-cells and B-cells T-cells show some staining, but B-cells as well (they should not show staining) IgG control: showed no stainig, thus secondary Ab is fine Ab: 5 ug/ml block w: none fix/perm: with commercialized Cytofix/perm solution

    Read More
    Answer

    Thank you for contacting us. Your credit note ID is xxx. I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. The credit note ID is for your reference only and does not automatically guarantee the credit. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

    Read More

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Perforin antibody [CB5.4]

     Excellent
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Lymphoid tissue)
    Specification
    Lymphoid tissue
    Fixative
    Zinc
    Antigen retrieval step
    None
    Permeabilization
    No
    Blocking step
    Rodent block-M (other company) as blocking agent for 20 minute(s) · Concentration: 100% · Temperature: 20°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jan 28 2009

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

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