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Perforin Monoclonal Antibody (eBioOMAK-D), PE, eBioscience™
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FIGURE: 1 / 23
Perforin Antibody (12-9392-82) in Flow
Intracellular staining of C57BL/6 splenocytes unstimulated (left) or stimulated with Mouse IL-2 Recombinant Protein (Product # 14-8021-64) (right) with Anti-Mouse CD49b (Integrin alpha 2) APC (Product # 17-5971-82) and 0.5 µg of Anti-Mouse Perforin PE using the Foxp3/Transcription Factor Staining Buffer Set (Product # 00-5523-00) and protocol. Quadrant lines demarcate isotype control staining.
Product Details
12-9392-82
Applications
Tested Dilution
Publications
Product Specifications
Species Reactivity
Mouse
Published species
Human,
Mouse
Host/Isotype
Rat
/ IgG2a, kappa
Recommended Isotype Control
Class
Monoclonal
Type
Antibody
Clone
eBioOMAK-D
Conjugate
Excitation/Emission Max
565/576 nm
View spectra
Form
Liquid
Concentration
0.2 mg/mL
Amount
100 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 intracellular staining and 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: 488-561 nm; Emission: 578 nm; Laser: Blue Laser, Green Laser, Yellow-Green 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; Perforin-1; Perforin1; PGFL; PIGF; PIGF-2; PLGF; pore forming protein; PRF1 (pore forming protein 1); RP11-710A11.3
Gene Aliases: Pfn; Pfp; Prf-1; Prf1
UniProt ID: (Mouse) P10820
Entrez Gene ID: (Mouse) 18646
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