Analyses of mTOR (pS2448) expression in stimulated human peripheral blood B cells. Left Panel: Flow cytometric analysis of mTOR (pS2448) expression. Human B lymphocytes were prepared from peripheral blood mononuclear cells (PBMC) by negative selection using the BD IMag™ Human B Lymphocyte Enrichment Set - DM (Cat. No. 558007). The B cells were serum starved overnight in culture and were either not stimulated (dashed line histogram) or were stimulated with Type C CpG oligonucleotide (1 μM, 4 hrs; InvivoGen, Cat. No. TLRL-2395). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655; 10 minutes at 37°C) and then permeabilized by adding BD Phosflow™ Perm Buffer III (Cat. No. 558050; 30 minutes on ice). The cells were washed twice with BD Pharmingen™ Stain Buffer (Cat. No. 554656) and then stained with BD Phosflow™ PE Mouse Anti-mTOR (pS2448) (Cat. No. 563489) and Alexa Fluor® 647 Mouse Anti-Human CD20 (Cat. No. 558054) antibodies using the BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from CD20 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD FACSCanto™ II Flow Cytometer. Right Panel: Western blot analysis of mTOR (pS2448) expression. Aliquots of the unstimulated (lane 1) or CPG-stimulated (lane 2) B cells described above were made into lysates. The lysates were electrophoresed, transferred to membranes and blotted using Purified Mouse Anti-mTOR (pS2448) antibody (Clone O21-404; 2 μg/mL). Phosphorylated mTOR (pS2448) was identified as ~240-245 kDa band.
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Analyses of mTOR (pS2448) expression in stimulated human peripheral blood B cells. Left Panel: Flow cytometric analysis of mTOR (pS2448) expression. Human B lymphocytes were prepared from peripheral blood mononuclear cells (PBMC) by negative selection using the BD IMag™ Human B Lymphocyte Enrichment Set - DM (Cat. No. 558007). The B cells were serum starved overnight in culture and were either not stimulated (dashed line histogram) or were stimulated with Type C CpG oligonucleotide (1 μM, 4 hrs; InvivoGen, Cat. No. TLRL-2395). The cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655; 10 minutes at 37°C) and then permeabilized by adding BD Phosflow™ Perm Buffer III (Cat. No. 558050; 30 minutes on ice). The cells were washed twice with BD Pharmingen™ Stain Buffer (Cat. No. 554656) and then stained with BD Phosflow™ PE Mouse Anti-mTOR (pS2448) (Cat. No. 563489) and Alexa Fluor® 647 Mouse Anti-Human CD20 (Cat. No. 558054) antibodies using the BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from CD20 positive-gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD FACSCanto™ II Flow Cytometer. Right Panel: Western blot analysis of mTOR (pS2448) expression. Aliquots of the unstimulated (lane 1) or CPG-stimulated (lane 2) B cells described above were made into lysates. The lysates were electrophoresed, transferred to membranes and blotted using Purified Mouse Anti-mTOR (pS2448) antibody (Clone O21-404; 2 μg/mL). Phosphorylated mTOR (pS2448) was identified as ~240-245 kDa band.
Product Details
BD Phosflow™
Mammalian target of rapamycin; FRAP1; RAFT1; RAPT1
Human (QC Testing), Mouse,Rat (Predicted)
Mouse IgG1, κ
Phosphorylated Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2736872
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO
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.
Product Notices
- 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).
- Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Alexa Fluor® 647 Mouse Anti-Human CD20 RUO
Size
50 Tests
Cat No.
558054
Human B Lymphocyte Enrichment Set - DM RUO
Cat No.
558007
563489 Rev. 1
Antibody Details
O21-404
The O21-404 monoclonal antibody recognizes the human mammalian target of rapmycin,(mTOR) that is phosphorylated at
serine residue 2448, mTOR (pS2448). mTOR belongs to the phosphoinositide-3-kinase (PI3K)-related (PIKK) family of kinases. mTOR is also known as mechanistic target of rapamycin (serine/threonine kinase), FRAP, RAFT1, and RAPT1. mTOR functions as an amino acid and ATP sensor to balance nutrient availability and cell growth. When nutrients are sufficiently available, mTOR is activated by phosphorylation at serine residue 2448 through the PI3 kinase/Akt signaling pathway. Phosphorylated mTOR in turn activates the p70 S6 kinase and contributes to the inactivation 4E-BP1, an elF4E inhibitor. In this way, mTOR participates in the upregulation of cellular protein synthesis, growth, proliferation and survival. mTOR function may be abnormally regulated in tumors.
563489 Rev. 1
Format Details
PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE 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 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
563489 Rev.1
Citations & References
Development References (6)
-
Brown EJ, Albers MW, Shin TB, et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994; 369(6483):756-758. (Biology). View Reference
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Chiu MI, Katz H, Berlin V. RAPT1, a mammalian homolog of yeast Tor, interacts with the FKBP12/rapamycin complex. Proc Natl Acad Sci U S A. 1994; 91(26):12574-12578. (Biology). View Reference
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Holz MK, Blenis J. Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase. J Biol Chem. 2005; 280(28):26089-26093. (Biology). View Reference
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Johnson SC, Rabinovitch PS, Kaeberlein M. mTOR is a key modulator of ageing and age-related disease. Nature. 2013; 493(7432):338-345. (Biology). View Reference
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Nave BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 1999; 344:427-431. (Biology). View Reference
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Sengupta S, Peterson TR, Sabatini DM. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell. 2010; 40(2):310-322. (Biology). View Reference
563489 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.
