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

  • 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) »
  • Lu J  et al. Nano-enabled pancreas cancer immunotherapy using immunogenic cell death and reversing immunosuppression. Nat Commun 8:1811 (2017). Read more (PubMed: 29180759) »
See all 16 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

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

Abcam user community

Verified customer

Submitted Jul 27 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
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

Dr. Varkha Agrawal

Verified customer

Submitted Jul 03 2013

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.

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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.

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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,

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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.

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Question
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.

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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.

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Abcam guarantees this product to work in the species/application used in this Abreview.
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

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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