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Invitrogen
Perforin Monoclonal Antibody (eBioOMAK-D), APC, eBioscience™
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FIGURE: 1 / 21
Perforin Antibody (17-9392-80) in Flow
Intracellular staining of C57BL/6 splenocytes cultured with recombinant mouse IL-2 for 4 days with Anti-Mouse CD49b (Integrin alpha 2) PE (Product # 12-5971-82) and 0.5 µg of Anti-Mouse Perforin APC using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. Cells in the lymphocyte gate were used for analysis.
Please note: We are reviewing Western blot images included in the antibody testing data in our catalog, including those provided by third parties. Unless expressly labeled or annotated as “raw-unedited”, Western blot images included in the antibody testing data in our catalog may have been edited, optimized or otherwise adjusted for presentation.
Product Details
17-9392-80
Applications
Tested Dilution
Publications
Product Specifications
Species Reactivity
Mouse
Published species
Mouse
Host/Isotype
Rat
/ IgG2a, kappa
Recommended Isotype Control
Class
Monoclonal
Type
Antibody
Clone
eBioOMAK-D
Conjugate
APC
APC
APC
Excitation/Emission Max
651/660 nm
View spectra
Form
Liquid
Concentration
0.2 mg/mL
Amount
25 µg
Purification
Affinity chromatography
Storage buffer
PBS, pH 7.2
Contains
0.09% sodium azide
Storage conditions
4°C, store in dark, DO NOT FREEZE!
Shipping conditions
Ambient (domestic); Wet ice (international)
RRID
Product Specific Information
Description: The eBioOMAK-D antibody reacts with mouse perforin (pore-forming protein, pfp, Prf). Perforin is one of the cytolytic mediators present in the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer cells (NK). Perforin is involved in the killing function by CTLs and NKs and has an important role in the immune response against tumors and virus infections.
By immunoblotting, eBioOMAK-D recognizes a ~70kDa band in lysates of CTLL-2 mouse cytotoxic cell line and in lysates of IL-2 stimulated but not unstimulated mouse splenocytes. By multi-color intracellular flow cytometric analysis, eBioOMAK-D staining is increased upon stimulation (IL-2 or anti-CD3/28). Intracellular flow staining results showing upregulation of protein expression have been confirmed by immunoblotting. Furthermore, stimulated Perforin Knock-out (developed by Walsh) splenocytes do not stain with eBioOMAK-D nor is any protein detectable by western blotting with eBioOMAK-D as well as other anti-mouse perforin antibodies. Please note that the Kagi perforin knock-out mice may synthesize a truncated form of the protein which may be recognized by eBioOMAK-D.
In IL-2 stimulated mouse splenocytes, NK cells (as determined by CD49b staining) contain perforin while CD8 cells contain little to none and can vary with culture conditions. This has been confirmed by staining and western blotting the two populations using both OMAK-D and P1-8 antibodies. In contrast stimulation of splenocytes with anti-CD3/CD28 antibodies does result in an increase of perforin on both NK cells and CD8 cells.
eBioOMAK-D is also cross-reactive to human perforin and co-stains CD56 positive cells in PBMC.
Expression of perforin and Granzyme B do not always correlate (as discussed above in the CD8 population of IL-2 stimulated splenocytes). Granzyme B typically is expressed earlier and at higher levels. Expression of Granzyme B is dramatically increased (more than 10,00 fold based on mRNA estimates and significantly at the protein level based on western blotting and flow analysis) compared to a minimal increase (10-100 fold) in perforin mRNA and protein with IL-2 stimulation.
For intracellular staining and flow cytometric analysis with direct conjugates of anti-mouse perforin, it is highly recommended to use the Foxp3 buffer system (Product # 00-5523). Other buffers may yield varying results.
Applications Reported: This eBioOMAK-D antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested: This eBioOMAK-D antibody has been tested by flow cytometric analysis of stimulated mouse splenocytes using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523) and protocol. Please refer to BestProtocols®: Protocol B: One step protocol for intracellular (nuclear) proteins. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest.
Excitation: 633-647 nm; Emission: 660 nm; Laser: Red Laser.
Filtration: 0.2 µm post-manufacturing filtered.
Target Information
Perforin is one of the major cytolytic proteins of cytolytic granules. Perforin is a cytolytic mediator and is stored in and released by cytoplasmic granules. Moreover, perforin is involved in immune defense against tumors and virus infections as mediated by cytotoxic lymphocytes. Perforin is a 555 amino acid protein with a 21 amino acid signal peptide, and has a molecular weight of 70 to 75 kD. Perforin is a pore forming protein with a mechanism of transmembrane channel formation similar to C9, and homology between perforin and C9 have been demonstrated. Studies show that perforin is expressed only in killer cell lines and not in helper T lymphocytes or other tumor cells tested. Perforin is known to be a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis. Defects in the perforin gene cause familial hemophagocytic lymphohistiocytosis type 2 (HPLH2), a rare and lethal autosomal recessive disorder of early childhood. Alternative splicing results in multiple transcript variants of perforin.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
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Bioinformatics
Protein Aliases: cytolysin; lymphocyte pore-forming protein; OMAK; OTTHUMP00000019759; P1; Perforin1; PGFL; PIGF; PIGF-2; PLGF; pore forming protein; PRF1 (pore forming protein 1); RP11-710A11.3
Gene Aliases: Pfn; Pfp; Prf-1
UniProt ID: (Mouse) P10820
Entrez Gene ID: (Mouse) 18646
immunological synapse formation
plasma membrane repair
leukocyte mediated cytotoxicity
T cell mediated cytotoxicity
defense response to tumor cell
immune response to tumor cell
positive regulation of immune response to tumor cell
circadian rhythm
protein secretion
protein import
ceramide biosynthetic process
protein homooligomerization
protein maturation
defense response to virus
positive regulation of killing of cells of other organism
protein transmembrane transport
granzyme-mediated programmed cell death signaling pathway
pyroptotic cell death
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