PE Mouse Anti-Human IFN-γ
Clone B27 (RUO)
- Brand BD Pharmingen™
- Alternative Name IFNG; Interferon-gamma; Interferon-γ; Type II interferon; MAF
- Vol. Per Test 20 µl
- Isotype Mouse IgG1, κ
- Reactivity Human (QC Testing) Rhesus, Baboon, Cynomolgus (Tested in Development)
Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Human IFN-γ Recombinant Protein
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
The B27 monoclonal antibody specifically binds to human interferon-γ (IFN-γ), a 14-18 kDa glycoprotein containing 143 amino acid residues. IFN-ã is a potent multifunctional cytokine produced by several activated cell types including NK, NKT, CD4+TCRáâ+, CD8+TCRáâ+, and TCRãä+ T cells. IFN-ã exerts its biological effects through specific binding to the high-affinity IFN-ã receptor complex comprised of IFN-ãRá (CD119) and IFN-ãRâ subunits. In addition to its antiviral effects, IFN-ã upregulates a number of lymphoid cell functions including the antimicrobial and anti-tumor responses of macrophages, NK cells, and neutrophils. In addition, IFN-γ influences the regulation of proliferation, differentiation, and effector responses of B cell and T cell subsets. These influences can involve IFN-ã's capacity to boost MHC class I and II expression by antigen-presenting cells as well as direct effects on B cells and T cells themselves. B27 is a neutralizing antibody. The use of B27 antibody for epitope mapping of human IFN-ã has been described. The B27 antibody has been reported not to bind to denatured IFN-ã.
- Format PE
- Excitation Source Blue 488 nm,Green 532 nm,Yellow/Green 561 nm
- Excitation Max 496 nm
- Emission Max 578 nm
R-phycoerythrin (PE) is an accessory photosynthetic pigment found in red algae. It exists in vitro as a 240-kDa protein with 23 phycoerythrobilin chromophores per molecule. This makes PE one of the brightest fluorochromes for flow cytometry applications, but its photobleaching properties make it unsuitable for fluorescence microscopy.
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Immunofluorescent Staining and Flow Cytometric Analysis: The PE-conjugated B27 antibody is useful for multicolor immunofluorescent staining and flow cytometric analysis to identify and enumerate IFN-γ producing cells within mixed cell populations (see image). This 100 Test Size formulation of the PE-conjugated B27 antibody has been pre-titrated to assure effective intracellular detection of human IFN-γ using 20 µl per 1 x 10e6 cells. For specific methodology, please visit our website, http://www.bdbiosciences.com/us/s/resources and refer to the protocols section under "Cytokines (Intracellular Staining)" or "Intracellular Flow".
A suitable mouse IgG1 isotype control for assessing the level of background staining on paraformaldehyde-fixed/saponinpermeabilized
human cells is also available in a 100 Test Size formulation PE-MOPC-21 (Cat. No. 559320). A useful control for demonstrating specificity of staining is the following: pre-block the paraformaldehyde-fixed/saponin-permeabilized cells with unlabeled B27 antibody (Cat. No. 554699/550011) prior to staining. The intracellular cytokine staining technique and blocking controls are described in detail by C. Prussin and D. Metcalfe.
Important Note: This pre-titered antibody solution does not contain a cell permeabilization agent. It is necessary to include a cell permeabilization agent when using the pre-titered antibody solution to stain fixed and permeabilized cells. Perm/Wash™ Buffer (Cat. No. 554723) contains the permeabilization agent saponin and is useful for this purpose as described in the USAGE section below.
1. Resuspend 1 x 10^6 fixed and permeabilized cells in 20 µl of the pre-titered antibody solution and 30 µl of 1X Perm/Wash™ Buffer.
2. Incubate the cell suspension for 15 minutes (4°C, in the dark).
3. Wash twice in 100 µl of 1X Perm/Wash™ Buffer.