Human mTOR ELISA Kit (HUFI01396)- High Sensitivity

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Human mTOR ELISA Kit
Product Code:	HUFI01396
Size:	96 Assays
Alias:	MTOR, Serine, threonine-protein kinase mTOR, Rapamycin target protein 1, Rapamycin target protein 1, RAPT1, Mechanistic target of rapamycin, Mammalian target of rapamycin, mTOR, FK506-binding protein 12-rapamycin complex-associated protein 1, FKBP12-rapamycin complex-associated protein, FRAP, FRAP1, FRAP2
Detection Method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human MTOR concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Human MTOR and the recovery rates were calculated by comparing the measured value to the expected amount of Human MTOR in samples.


Matrix	Recovery Range (%)	Average (%)
serum(n=5)	90-101	97
EDTA plasma(n=5)	85-99	94
UFH plasma(n=5)	85-104	94

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human MTOR and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.


Sample	1:2	1:4	1:8
serum(n=5)	86-96%	90-103%	85-103%
EDTA plasma(n=5)	82-99%	86-98%	86-101%
UFH plasma(n=5)	88-99%	82-100%	80-100%

CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials 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

Protein Information

Heading 1	Heading 2
UniProt	P42345
NCBI GenInfo Identifier	1169735
NCBI Gene ID	2475
Molecular Weight	288,892 Da
Protein Family	Serine/threonine-protein kinase

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Type

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.

mTOR Background

Mammalian Target of Rapamycin

mTOR, short for mammalian target of rapamycin, is a crucial protein kinase that plays a pivotal role in regulating cell growth, proliferation, and metabolism. It serves as a central hub for integrating various signals, including growth factors, nutrients, and energy status, to coordinate cellular responses. By sensing the availability of these factors, mTOR controls essential cellular processes such as protein synthesis, autophagy, and cell cycle progression. Dysregulation of the mTOR signaling pathway has been implicated in numerous diseases, including cancer, metabolic disorders, and neurodegenerative conditions. Understanding the activity of mTOR and its downstream targets is essential for unraveling the underlying mechanisms of these diseases and developing potential therapeutic interventions.

mTOR Structure

mTOR, or mammalian target of rapamycin, is a large serine/threonine protein kinase with a molecular weight of approximately 289 kDa. It is composed of multiple functional domains that contribute to its complex structure and diverse cellular functions. The primary structure of mTOR includes the N-terminal HEAT repeat domains, which are involved in protein-protein interactions, and the C-terminal kinase domain responsible for its enzymatic activity. Within the kinase domain, there are key regulatory regions such as the FAT, FATC, and FRB domains that control mTOR's activation and interaction with other proteins and signaling molecules. The intricate structure of mTOR enables it to interact with various upstream regulators and downstream effectors, allowing for precise modulation of cellular processes involved in growth, metabolism, and autophagy.

mTOR Activation

mTOR activation is tightly regulated and can be influenced by various factors. One of the well-known activators of mTOR is a compound called rapamycin. Rapamycin functions by binding to a protein called FKBP12, forming a complex that inhibits the activity of mTOR complex 1 (mTORC1). However, there are also other activators that stimulate mTOR signaling, such as growth factors (e.g., insulin and insulin-like growth factor-1) and nutrients (e.g., amino acids, glucose). These activators promote the phosphorylation and activation of mTOR, leading to the initiation of downstream signaling cascades that regulate key cellular processes. Understanding the mechanisms of mTOR activation and identifying specific activators can provide valuable insights into modulating mTOR signaling and potentially developing therapeutic strategies for diseases associated with dysregulated mTOR activity.

mTOR ELISA Kit FAQs

What is the mTOR ELISA Kit used for?

The mTOR ELISA kit allows researchers and clinicians to assess and quantify mTOR protein levels, providing valuable insights into the regulation and dysregulation of this important signaling pathway in various diseases and biological contexts.

What are the advantages of using the mTOR ELISA Kit?

The mTOR ELISA Kit offers several advantages, including high sensitivity, accuracy, and reproducibility. It provides a user-friendly and reliable method to quantify mTOR levels in biological specimens, allowing for precise measurements and robust data analysis.

What sample types are compatible with mTOR ELISA Kit?

The mTOR ELISA Kit is compatible with various sample types, including serum, plasma, cell lysates, and tissue homogenates. It provides flexibility in sample selection, allowing researchers to analyze mTOR levels in different biological matrices.

What are the storage requirements with mTOR ELISA Kit?

The mTOR ELISA Kit components should be stored according to the instructions provided in the kit manual. Generally, it is recommended to store the kit components at the recommended temperature to ensure their stability and optimal performance.

What should I do if my assay results are not optimal?

If you encounter any issues or have suboptimal assay results, we recommend contacting our dedicated support team for assistance. They will be available to provide troubleshooting guidance, answer your questions, and ensure you achieve the best possible results with the mTOR ELISA Kit.

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Human mTOR ELISA Kit

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