Flow cytometric (Panels 1a and 1b) and Western blot (Panel 1c) analyses of Bcl-2 (pS70) expressed by Jurkat cells. Jurkat cells were either not treated (Panel 1a, dashed line histogram; Panel 1c, C) or treated (Panel 1a, solid line histogram; Panel 1b; Panel 1c, T) with 100 nM Taxol (Paclitaxel; Sigma, Cat. No. T7191; 24 hr, 37°C). For Panel 1a, cells were fixed in BD Phosflow™ Cytofix Buffer (Cat. No. 554655; 10 min, 37˚C) and permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050; 30 min, on ice) prior to staining with BD Phosflow™ PE-CF594 Mouse Anti-Bcl-2 (pS70) (Cat. No. 562679). For optimal co-staining of total cellular DNA (Panel 1b), cells were fixed and permeabilized in 70% ethanol (30 min, on ice) prior to staining with DAPI (Sigma, Cat. No. D8417) and PE-CF594 Mouse Anti-Bcl-2 (pS70). Histograms (Panel 1a) and a two-color dot plot showing DNA (DAPI) versus Bcl-2 (pS70) levels (Panel 1b) were generated from gated events with the light scatter characteristics of intact cells using a BD™ LSR II Flow Cytometer System. For Western blot (Panel 1c), cell lysates (15 µg total cell protein/lane) were blotted using Purified Mouse Anti-Bcl-2 (pS70) (Cat. No. 562529; 0.125, 0.063, 0.032 µg/ml for Lanes 1, 2, and 3, respectively). Bcl-2 (pS70) was identified as a ~28 kDa band. Flow cytometric (Panels 2a and 2b) and Western blot (Panel 2c) analyses of Bcl-2 (pS70) expressed by human peripheral blood mononuclear cells. PHA-stimulated (Sigma Cat. No. L1668; 20 μg/ml for 3 days;) PBMC were either not treated (Panel 2a, dashed line histogram; Panel 2c, C) or treated (Panel 2a, solid line histogram; Panel 2b; Panel 2c, T) with Taxol (100 nM, 24 hr, 37°C). Flow cytometric analyses of Bcl-2 (pS70) expression without (Panel 2a) or with (Panel 2b) co-staining for DNA content were performed using a BD LSRFortessa™ Cell Analyzer System. Lysates from 1X10^6 PBMC were blotted using Purified Mouse Anti-Bcl-2 (pS70) antibody (2.0 µg/ml) as described above.
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Flow cytometric (Panels 1a and 1b) and Western blot (Panel 1c) analyses of Bcl-2 (pS70) expressed by Jurkat cells. Jurkat cells were either not treated (Panel 1a, dashed line histogram; Panel 1c, C) or treated (Panel 1a, solid line histogram; Panel 1b; Panel 1c, T) with 100 nM Taxol (Paclitaxel; Sigma, Cat. No. T7191; 24 hr, 37°C). For Panel 1a, cells were fixed in BD Phosflow™ Cytofix Buffer (Cat. No. 554655; 10 min, 37˚C) and permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050; 30 min, on ice) prior to staining with BD Phosflow™ PE-CF594 Mouse Anti-Bcl-2 (pS70) (Cat. No. 562679). For optimal co-staining of total cellular DNA (Panel 1b), cells were fixed and permeabilized in 70% ethanol (30 min, on ice) prior to staining with DAPI (Sigma, Cat. No. D8417) and PE-CF594 Mouse Anti-Bcl-2 (pS70). Histograms (Panel 1a) and a two-color dot plot showing DNA (DAPI) versus Bcl-2 (pS70) levels (Panel 1b) were generated from gated events with the light scatter characteristics of intact cells using a BD™ LSR II Flow Cytometer System. For Western blot (Panel 1c), cell lysates (15 µg total cell protein/lane) were blotted using Purified Mouse Anti-Bcl-2 (pS70) (Cat. No. 562529; 0.125, 0.063, 0.032 µg/ml for Lanes 1, 2, and 3, respectively). Bcl-2 (pS70) was identified as a ~28 kDa band. Flow cytometric (Panels 2a and 2b) and Western blot (Panel 2c) analyses of Bcl-2 (pS70) expressed by human peripheral blood mononuclear cells. PHA-stimulated (Sigma Cat. No. L1668; 20 μg/ml for 3 days;) PBMC were either not treated (Panel 2a, dashed line histogram; Panel 2c, C) or treated (Panel 2a, solid line histogram; Panel 2b; Panel 2c, T) with Taxol (100 nM, 24 hr, 37°C). Flow cytometric analyses of Bcl-2 (pS70) expression without (Panel 2a) or with (Panel 2b) co-staining for DNA content were performed using a BD LSRFortessa™ Cell Analyzer System. Lysates from 1X10^6 PBMC were blotted using Purified Mouse Anti-Bcl-2 (pS70) antibody (2.0 µg/ml) as described above.
제품 세부 정보
BD Phosflow™
Hu Bcl-2 (pS70) PE-CF594 N46-467 50Tst
BCL2; Apoptosis regulator Bcl-2; B-cell CLL/lymphoma 2; PPP1R50
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1
Phosphorylated Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2737719
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO
준비 및 보관
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 BD Horizon™ PE-CF594 under optimum conditions, and unconjugated antibody and free PE-CF594 were removed.
제품 고시
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- 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.
- Texas Red is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- CF™ is a trademark of Biotium, Inc.
- When excited by the yellow-green (561-nm) laser, the fluorescence may be brighter than when excited by the blue (488-nm) laser.
- This product is provided under an Agreement between BIOTIUM and BD Biosciences. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications owned or licensed by Biotium, Inc. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
- Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using multi-laser cytometers, which may directly excite both PE and CF™594.
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- All other brands are trademarks of their respective owners.
관련 제품
Stain Buffer (FBS) RUO
사이즈
500 mL
카탈로그 No
554656
Fixation Buffer RUO
사이즈
100 mL
카탈로그 No
554655
Perm Buffer III RUO
사이즈
125 mL
카탈로그 No
558050
Purified Mouse anti-Human Bcl-2 (pS70) RUO
사이즈
0.1 mg
카탈로그 No
562529
562679 Rev. 1
항체 세부 정보
N46-467
The N46-467 monoclonal antibody specifically binds to Bcl-2 (pS70), ie, the Bcl-2 protein phosphorylated at the Ser70 site. Bcl-2 is a ~ 26 kDa intracellular, integral membrane protein found primarily in the nuclear envelope, endoplasmic reticulum and outer mitochondrial membrane. Bcl-2 is encoded by the BCL2 (B-cell CLL/lymphoma 2) gene and is also known as Apoptosis regulator Bcl-2. Members of the Bcl-2 family play a major role in regulating the response of cells to apoptotic signals. Bcl-2 is one of the anti-apoptotic members of the Bcl-2 family. Bcl-2 knockout mice showed pronounced lymphoid apoptosis and other apoptosis related lesions later in life. Bcl-2 is a proto-oncogene because it blocks apoptosis and provides a selective survival advantage in many cell types and thus contributes to tumorigenesis. It has been implicated in several types of cancers, such as breast, prostate, and melanoma . Bcl-2 contains multiple phosphorylation sites including Thr56, Ser70, Thr74 and Ser87. Phosphorylation of Bcl-2 Ser70 has been shown to be a mitotic marker. Phosphorylation at this site regulates Bcl-2's anti-apoptotic activity and has recently been implicated in promoting autophagy. Several studies have shown that Bcl-2 phosphorylation is caused by c-Jun N-terminal kinase (JNK).
This antibody is conjugated to BD Horizon™ PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red®. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red® yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red® (eg 610/20-nm filter).
562679 Rev. 1
형광 세부 정보
PE-CF594
BD Horizon™ PE-CF594 dye is a part of the BD PE family of dyes. This tandem fluorochrome is comprised of a R-Phycoerythrin (PE) donor that has excitation maxima (Ex Max) of 496-nm and 566-nm and an acceptor dye with an emission maximum (Em Max) at 615-nm. PE-CF594, driven by BD innovation, is designed to be excited by the blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and detected using an optical filter centered near 615 nm (e.g., a 610/20-nm bandpass filter). The donor dye can be excited by the Blue (488-nm), Green (532-nm) and yellow-green (561-nm) lasers and the acceptor dye can be excited by the green (532-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE-CF594
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
615 nm
562679 Rev.1
인용 및 참고 문헌
개발 참고 자료 (6)
-
Deng X, Xiao L, Lang W, et al. Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem. 2001; 276(26):23681-23688. (Biology). 참조 보기
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Geng F, Tang L, Li Y, et al. Allyl isothiocyanate arrests cancer cells in mitosis, and mitotic arrest in turn leads to apoptosis via Bcl-2 protein phosphorylation. J Biol Chem. 2011; 286(37):32259-32267. (Biology). 참조 보기
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Ling YH, Tornos C, Perez-Soler R. Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis. J Biol Chem. 1998; 273(30):18984-18991. (Biology). 참조 보기
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Maundrell K, Antonsson B, Magnenat E, et al. Bcl-2 undergoes phosphorylation by c-Jun N-terminal kinase/stress-activated protein kinases in the presence of the constitutively active GTP-binding protein Rac1. J Biol Chem. 1997; 272(40):25238-42. (Biology). 참조 보기
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Pattingre S, Bauvy C, Carpentier S, Levade T, Levine B, Codogno P.. Role of JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy. J Biol Chem. 2009; 284(5):2719-2728. (Biology). 참조 보기
-
Sarkar S, Korolchuk VI, Renna M, et al. Complex inhibitory effects of nitric oxide on autophagy. Mol Cell. 2011; 43(1):19-32. (Biology). 참조 보기
562679 Rev. 1
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
