D267-3 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures. MONOCLONAL ANTIBODY CD267/TACI Code No. Clone Subclass Quantity Concentration D267-3 11H3 Mouse IgG2a 100 g 1 mg/mL BACKGROUND: CD267, also known as TACI SPECIES CROSS REACTIVITY: (transmembrane activator and calcium modulator and Species Human Mouse Rat cyclophilin ligand interactor) or TNFRSF13B, is a member of the tumor necrosis factor (TNF) receptor family that Cell transfectant Not Tested Not Tested serves as a receptor for B-cell activating factor of the TNF family (BAFF/CD257) and as a proliferation-inducing Reactivity on FCM + ligand (APRIL/CD256). BAFF and APRIL bind both TACI and BCMA (B cell maturation Ag/CD269), with BAFF additionally and exclusively binding the BAFF receptor INTENDED USE: (BAFF-R/CD268) while APRIL can also bind to heparin For Research Use Only. Not for use in diagnostic procedures. sulfate proteoglycans (HSPG). TACI is expressed on B cells and activated T cells. BCMA is exclusively expressed on B cells, whereas BAFF-R is expressed mainly on B cells REFERENCES: but also on T cells. It is reported that mutations of 1) Sakurai, D., et al., Blood 109, 2961-2967 (2007) CD267/TACI are showed in patients with IgA deficiency 2) Hase, H., et al., Blood 103, 2257-2265 (2004) and common variable immunodeficiency (CVID). Sakurai Clone 11H3 is used in reference number 1). et al., reported that TACI positively regulated APRIL-induced IgA production in collaboration with HSPG and TACI negatively regulated BAFF-induced B-cell proliferation and production of IgA and IgG. SOURCE: This antibody was purified from hybridoma (clone 11H3) supernatant using protein A agarose. This hybridoma was established by fusion of mouse myeloma cell NS-1 with C57BL/6 mouse splenocyte immunized with human TACI full-length cDNA-transfected cells. FORMULATION: 100 g IgG in 100 L volume of PBS containing 50% glycerol, pH 7.2. No preservative is Flow cytometric analysis of CD267 contained. expression on transfectant. Open histogram indicates the reaction of isotypic control to the cells. Shaded histogram STORAGE: This antibody solution is stable for one year indicates the reaction of D267-3 to the cells. from the date of purchase when stored at -20oC. REACTIVITY: This antibody reacts with CD267 antigen on Flow cytometry. Clone 11H3 has an agonistic activity and it doesn’t block binding of BAFF to TACI. APPLICATIONS: Western blotting; Not tested Immunoprecipitation; Not tested Immunohistochemistry; Not tested Immunocytochemistry; Not tested Flow cytometry; 10 g/mL (final concentration) Flow cytometric analysis of CD267 Detailed procedure is provided in the following expression on EBV-LCL cells. Shaded PROTOCOL. histogram indicates the reaction of isotypic control to the cells. Open histogram indicates the reaction of D267-3 to the cells. MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp e-mail [email protected], TEL 052-238-1904 D267-3 Page 2 of 2 PROTOCOL: D222-4 FITC labeled anti-mouse GITR (DTA-1) Flow cytometric analysis for floating cells D222-5 PE labeled anti-mouse GITR (DTA-1) We usually use Fisher tubes or equivalents as reaction tubes K0031-3 anti-HVEM (122) for all steps described below. K0031-4 FITC labeled anti-HVEM (122) 1) Wash the cells 3 times with washing buffer [PBS MD-10-3 anti-human Fas (CD95) (UB2) MD-10-4 FITC labeled anti-human Fas (CD95) (UB2) containing 2% fetal calf serum (FCS) and 0.1% NaN3]. 2) Resuspend the cells with washing buffer (5 x 106 MD-10-5 PE labeled anti-human Fas (CD95) (UB2) cells/mL). MD-11-3 anti-human Fas (CD95) (ZB4) 3) Add 50 L of the cell suspension into each tube, and SY-001 anti-human Fas (CD95) (CH-11) centrifuge at 500 x g for 1 minute at room temperature D041-3 anti-human FasL (CD178) (4H9) (20~25oC). Remove supernatant by careful aspiration. D041-4 FITC labeled anti-human FasL (CD178) (4H9) D041-5 PE labeled anti-human FasL (CD178) (4H9) 4) Add 20 L of Clear Back (human Fc receptor blocking reagent, MBL; code no. MTG-001) to the cell pellet after D041-6 Biotin labeled anti-human FasL (CD178) (4H9) tapping. Mix well and incubate for 5 minutes at room D042-3 anti-human FasL (CD178) (4A5) temperature. D026-3 anti-mouse Fas (CD95) (RMF2) D027-3 anti-mouse Fas (CD95) (RMF6) 5) Add 40 L of the primary antibody at the concentration as D057-3 anti-mouse FasL (CD178) (FLIM58) suggest in the APPLICATIONS diluted in the washing D057-4 FITC labeled anti-mouse FasL (CD178) (FLIM58) buffer. Mix well and incubate for 30 minutes at room D057-6 Biotin labeled anti-mouse FasL (CD178) (FLIM58) temperature. D069-3 anti-mouse FasL (CD178) (FLIM4) 6) Add 1 mL of the washing buffer followed by M031-3 anti-TRADD (3E11) centrifugation at 500 x g for 1 minute at room temperature. K0033-3 anti-DR3 (B65) Remove supernatant by careful aspiration. K0033-4 FITC labeled anti-DR3 (B65) 7) Add 30 L of 1:100 FITC conjugated anti-mouse IgG M112-3 anti-mouse TRAF2 (6F8) (MBL; code no. 238) diluted with the washing buffer. Mix 592 anti-mouse TRAF2 (polyclonal) well and incubate for 30 minutes at room temperature. 597 anti-mouse TRAF6 (polyclonal) 8) Add 1 mL of the washing buffer followed by M092-3 anti-TRAF6 (1F8) centrifugation at 500 x g for 1 minute at room temperature. Remove supernatant by careful aspiration. 9) Resuspend the cells with 500 L of the washing buffer and analyze by a flow cytometer. (Positive controls for Flow cytometry; transfectant, EBV-LCL) RELATED PRODUCTS: D200-3 CD257/BAFF/BLyS (1D6) D200-4 FITC labeled CD257/BAFF/BLyS (1D6) D201-3 CD268/BAFFR/BR3 (8A7) D201-4 FITC labeled CD268/BAFFR/BR3 (8A7) D113-3 anti-human TNF (#1) D114-3 anti-human TNF (#1) K0039-3 CD120a/TNFR1 (H398) K0039-4 FITC labeled CD120a/TNFR1 (H398) K0040-3 CD120b/TNFR2 (80M2) K0040-4 FITC labeled CD120b/TNFR2 (80M2) K0040-5 PE labeled CD120b/TNFR2 (80M2) K0145-3 CD30 (Ber-H2) K0145-4 FITC labeled CD30 (Ber-H2) K0029-3 CD137 (4B4-1) K0029-4 FITC labeled CD137 (4B4-1) K0030-3 CD137L (5F4) K0030-4 FITC labeled CD137L (5F4) D125-3 CD134/OX40 (W4-3) D126-3 CD252/OX40L (TAG-34) D051-3 CD154 (5F3) D051-4 FITC labeled CD154 (5F2) D222-3 anti-mouse GITR (DTA-1) D222-3M2 anti-GITR (DTA-1)(functional grade) 080523-1.1 .