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Expression of cell surface IFN-γRβ chains by BALB/c splenic lymphocytes (Left panel) and mouse A20 cells (Right Panel). BALB/c spleen cells and A20 cells (mouse B cell lymphoma) were preincubated (~15 min., 4°C) with purified 2.4G2 antibody [rat anti-mouse CD16 (FcγIII)/CD32 (FcγII); Cat. No. 553142; 1 µg antibody/10e6 cells] to block nonspecific staining due to immunoglobulin Fc receptors. The cells were stained (45 min., 4°C) with purified MOB-47 antibody (either at 0.5 µg mAb/10e6 BALB/c cells or at 0.125 µg mAb/10e6 A20 cells; Cat. No. 559917). After washing, the cells were incubated (30 min., 4°C) with a biotin-conjugated cocktail of mouse anti-hamster antibodies (Clones G70-204 + G94-56; Cat. No. 554010; 0.5 µg mAb cocktail/10e6 cells). Finally, the cells were washed and incubated with R-PE-conjugated streptavidin (Cat. No. 554061; 0.015 µg PE-SA/10e6 cells). After washing, the cells were analyzed with a FACScan™ Flow Cytometer. The immunofluorescent staining patterns for splenic lymphocytes (left panel) and A20 cells (right panel) that were either not stained with MOB-47 (grey line) or were stained with purified MOB-47 (black line) followed by the 2nd and 3rd layer reagents. The overlapping histograms were generated from reanalyzed flow cytometric data files that were gated for cells that had the light-scattering characteristics of lymphocytes (left panel) or viable tumor cells (right panel).
BD Pharmingen™ Purified Hamster Anti-Mouse IFN-γ Receptor β Chain
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준비 및 보관
권장 분석 절차
Immunofluorescent Staining and Flow Cytometric Analysis: The purified form of MOB-47 (Cat. No. 559917) can be used for the immunofluorescent staining (≤ 1 µg antibody/10e6 cells) and flow cytometric analysis of normal mouse cells or cell lines to measure their expressed levels of IFN-γRβ. An appropriate purified immunoglobulin isotype control is clone A19-3 (Cat. No. 553969). A three-layer staining protocol is recommended for maximizing the detection IFN-γRβ chains expressed by cells as detailed in the figure legend.
Note: The IFN-γRβ receptor epitope recognized by the MOB-47 antibody is blocked by bound IFN-γ. Thus, it has been reported that cells which have bound IFN-γ can be pretreated with dilute acid to remove the bound ligand before they are stained.
IP/WB: The MOB-47 antibody has been reported to be useful for the immunoprecipitation and Western blotting of IFN-γRβ chains from lysates of cloned mouse T cells.
제품 고시
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- Although hamster immunoglobulin isotypes have not been well defined, BD Biosciences Pharmingen has grouped Armenian and Syrian hamster IgG monoclonal antibodies according to their reactivity with a panel of mouse anti-hamster IgG mAbs. A table of the hamster IgG groups, Reactivity of Mouse Anti-Hamster Ig mAbs, may be viewed at http://www.bdbiosciences.com/documents/hamster_chart_11x17.pdf.
The MOB-47 antibody recognizes the 60-65 kD beta chain subunit of the mouse interferon-γ receptor (IFN-γRβ). The IFN-γ receptor β chain is expressed by a variety of normal mouse cell types and cell lines that are responsive to IFN-γ including T and B lymphocytes, NK cells, monocytes, macrophages, granulocytes and fibroblasts. The IFN-γRβ receptor epitope recognized by the MOB-47 antibody is blocked by bound IFN-γ. The immunogen used to generate this hybridoma was the purified soluble extracellular domain of the mouse IFN-γRβ chain protein.
개발 참고 자료 (1)
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Bach EA, Szabo SJ, Dighe AS, et al. Ligand-induced autoregulation of IFN-gamma receptor beta chain expression in T helper cell subsets.. Science. 1995; 270(5239):1215-8. (Clone-specific: Flow cytometry, Immunoprecipitation, Western blot). 참조 보기
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