Product Name:	Rat Serine/threonine-protein kinase mTOR (Mtor) ELISA Kit
SKU:	RTEB1022
Size:	96T
Target:	Rat Serine/threonine-protein kinase mTOR (Mtor)
Synonyms:	FK506-binding protein 12-rapamycin complex-associated protein 1, FKBP12-rapamycin complex-associated protein, Mammalian target of rapamycin, Mechanistic target of rapamycin, Rapamycin target protein 1, mTOR, RAPT1, Frap1, Raft1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/ml
Sensitivity:	0.08ng/mL

Intra CV:

6.9%

Inter CV:

9.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	91-101%	105-117%	105-115%	100-109%
EDTA Plasma(N=5)	100-109%	92-102%	106-116%	100-110%
Heparin Plasma(N=5)	106-115%	105-114%	102-111%	111-120%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	109	103-115
Plasma	111	105-117

Function:

Serine/threonine protein kinase which is a central regulator of cellular metabolism, growth and survival in response to hormones, growth factors, nutrients, energy and stress signals. MTOR directly or indirectly regulates the phosphorylation of at least 800 proteins. Functions as part of 2 structurally and functionally distinct signaling complexes mTORC1 and mTORC2 (mTOR complex 1 and 2). Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. This includes phosphorylation of EIF4EBP1 and release of its inhibition toward the elongation initiation factor 4E (eiF4E). Moreover, phosphorylates and activates RPS6KB1 and RPS6KB2 that promote protein synthesis by modulating the activity of their downstream targets including ribosomal protein S6, eukaryotic translation initiation factor EIF4B, and the inhibitor of translation initiation PDCD4. Stimulates the pyrimidine biosynthesis pathway, both by acute regulation through RPS6KB1-mediated phosphorylation of the biosynthetic enzyme CAD, and delayed regulation, through transcriptional enhancement of the pentose phosphate pathway which produces 5-phosphoribosyl-1-pyrophosphate (PRPP), an allosteric activator of CAD at a later step in synthesis, this function is dependent on the mTORC1 complex. Regulates ribosome synthesis by activating RNA polymerase III-dependent transcription through phosphorylation and inhibition of MAF1 an RNA polymerase III-repressor. In parallel to protein synthesis, also regulates lipid synthesis through SREBF1/SREBP1 and LPIN1. To maintain energy homeostasis mTORC1 may also regulate mitochondrial biogenesis through regulation of PPARGC1A. mTORC1 also negatively regulates autophagy through phosphorylation of ULK1. Under nutrient sufficiency, phosphorylates ULK1 at 'Ser-758', disrupting the interaction with AMPK and preventing activation of ULK1. Also prevents autophagy through phosphorylation of the autophagy inhibitor DAP. mTORC1 exerts a feedback control on upstream growth factor signaling that includes phosphorylation and activation of GRB10 a INSR-dependent signaling suppressor. Among other potential targets mTORC1 may phosphorylate CLIP1 and regulate microtubules. As part of the mTORC2 complex MTOR may regulate other cellular processes including survival and organization of the cytoskeleton. Plays a critical role in the phosphorylation at 'Ser-473' of AKT1, a pro-survival effector of phosphoinositide 3-kinase, facilitating its activation by PDK1. mTORC2 may regulate the actin cytoskeleton, through phosphorylation of PRKCA, PXN and activation of the Rho-type guanine nucleotide exchange factors RHOA and RAC1A or RAC1B. mTORC2 also regulates the phosphorylation of SGK1 at 'Ser-421'. Regulates osteoclastogensis by adjusting the expression of CEBPB isoforms.

Uniprot:	P42346
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Serine/threonine-protein kinase mTOR
Sub Unit:	Part of the mammalian target of rapamycin complex 1 (mTORC1) which contains MTOR, MLST8, RPTOR, AKT1S1/PRAS40 and DEPTOR. The mTORC1 complex is a 1 Md obligate dimer of two stoichiometric heterotetramers with overall dimensions of 290 A x 210 A x 135 A. It has a rhomboid shape and a central cavity, the dimeric interfaces are formed by interlocking interactions between the two MTOR and the two RPTOR subunits. The MLST8 subunit forms distal foot-like protuberances, and contacts only one MTOR within the complex, while the small PRAS40 localizes to the midsection of the central core, in close proximity to RPTOR. Part of the mammalian target of rapamycin complex 2 (mTORC2) which contains MTOR, MLST8, PRR5, RICTOR, MAPKAP1 and DEPTOR. Interacts with PRR5 and RICTOR; the interaction is direct within the mTORC2 complex. Interacts with WAC; WAC positively regulates MTOR activity by promoting the assembly of the TTT complex composed of TELO2, TTI1 and TTI2 and the RUVBL complex composed of RUVBL1 and RUVBL2 into the TTT-RUVBL complex which leads to the dimerization of the mTORC1 complex and its subsequent activation. Interacts with PLPP7 and PML. Interacts with UBQLN1. Interacts with TTI1 and TELO2. Interacts with CLIP1; phosphorylates and regulates CLIP1. Interacts with NBN. Interacts with BRAT1.
Research Area:	Cancer
Subcellular Location:	Endoplasmic reticulum membrane Peripheral membrane protein Cytoplasmic side Golgi apparatus membrane Peripheral membrane protein Cytoplasmic side Mitochondrion outer membrane Peripheral membrane protein Cytoplasmic side Lysosome Cytoplasm Nucleus PML body Shuttles between cytoplasm and nucleus. Accumulates in the nucleus in response to hypoxia (By similarity). Targeting to lysosomes depends on amino acid availability and RRAGA and RRAGB (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	mTOR: an atypical kinase belonging to the PIKK family of kinases. Is the catalytic subunit of two protein complexes, mTORC1 and mTORC2. mTORC1 activates S6K and inactivates 4E-BP1, up-regulating protein synthesis. mTORC1 contains Raptor, a positive regulatory subunit and scaffold for recruiting substrates, two negative regulators, PRAS40 and DEPTOR, and mLST8; it is a target for the cell-cycle arrest and immunosuppressive effects of the FKBP12-rapamycin complex. mTORC2, a downstream effector of PI3K, is insensitive to rapamycin and activates Akt by phosphorylating a key activation site. mTORC2 contains regulatory subunits Rictor and mSIN1, PROTOR, mLST8, and the negative regulator DEPTOR. mTORC1 suppresses PI3K activity via a strong negative feedback loop that involves S6K1. Inhibiting mTORC1 ablates this negative feedback loop and potentiates PI3K signaling. Known inhibitors of mTOR include rapamycin, temsirolimus (CCI-779).
UniProt Protein Details:	Protein type:ATYPICAL group; Autophagy; EC 2.7.11.1; FRAP subfamily; Kinase, protein; Motility/polarity/chemotaxis; PIKK family; Protein kinase, Ser/Thr (non-receptor); Protein kinase, atypical Chromosomal Location of Human Ortholog: 5q36 Cellular Component: cell soma; cytoplasm; cytosol; dendrite; endomembrane system; lysosomal membrane; lysosome; membrane; nucleus; PML body; TORC2 complex Molecular Function:kinase activity; phosphoprotein binding; protein binding; protein domain specific binding; protein kinase activity; protein kinase binding; protein serine/threonine kinase activity; ribosome binding Biological Process: 'de novo' pyrimidine base biosynthetic process; brain development; cardiac cell development; cardiac muscle cell development; cardiac muscle contraction; cardiac muscle development; cell aging; cell growth; cell projection organization and biogenesis; cellular response to amino acid starvation; cellular response to nutrient levels; energy reserve metabolic process; germ cell development; heart morphogenesis; long-term memory; maternal process involved in pregnancy; mRNA stabilization; multicellular organism growth; negative regulation of autophagy; negative regulation of cell size; negative regulation of macroautophagy; negative regulation of muscle atrophy; negative regulation of NFAT protein import into nucleus; negative regulation of protein amino acid phosphorylation; negative regulation of protein ubiquitination; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; phosphorylation; positive regulation of actin filament polymerization; positive regulation of endothelial cell proliferation; positive regulation of keratinocyte migration; positive regulation of lipid biosynthetic process; positive regulation of neurogenesis; positive regulation of neuron maturation; positive regulation of nitric oxide biosynthetic process; positive regulation of oligodendrocyte differentiation; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of protein amino acid phosphorylation; positive regulation of protein kinase B signaling cascade; positive regulation of smooth muscle cell proliferation; positive regulation of stress fiber formation; positive regulation of transcription from RNA polymerase III promoter; positive regulation of translation; post-embryonic development; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of actin cytoskeleton organization and biogenesis; regulation of carbohydrate metabolic process; regulation of carbohydrate utilization; regulation of cell size; regulation of fatty acid beta-oxidation; regulation of glycogen biosynthetic process; regulation of GTPase activity; regulation of myelination; regulation of osteoclast differentiation; regulation of protein amino acid phosphorylation; regulation of protein kinase activity; regulation of protein kinase B signaling cascade; regulation of response to food; response to amino acid stimulus; response to cocaine; response to insulin stimulus; response to morphine; ruffle organization and biogenesis; social behavior; spinal cord development; TOR signaling pathway; visual learning; voluntary musculoskeletal movement; wound healing
NCBI Summary:	binds the complex formed by the immunosuppressive drug rapamycin and its receptor FKBP12; may play a role in the cell cycle G1 to S transition [RGD, Feb 2006]
UniProt Code:	P42346
NCBI GenInfo Identifier:	1169736
NCBI Gene ID:	56718
NCBI Accession:	P42346.1
UniProt Related Accession:	P42346
Molecular Weight:	288,794 Da
NCBI Full Name:	Serine/threonine-protein kinase mTOR
NCBI Synonym Full Names:	mechanistic target of rapamycin
NCBI Official Symbol:	Mtor
NCBI Official Synonym Symbols:	Frap1; RAFT1
NCBI Protein Information:	serine/threonine-protein kinase mTOR
UniProt Protein Name:	Serine/threonine-protein kinase mTOR
UniProt Synonym Protein Names:	FK506-binding protein 12-rapamycin complex-associated protein 1; FKBP12-rapamycin complex-associated protein; Mammalian target of rapamycin; mTOR; Mechanistic target of rapamycin; Rapamycin target protein 1; RAPT1
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	Mtor

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Rat Serine/threonine-protein kinase mTOR (Mtor) ELISA Kit (RTEB1022)

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