Revision 2

#57002

Store at +4C

PathScan® RP Phospho-TFEB (Ser211) Sandwich ELISA Kit

1 Kit

(96 assays)

Species Cross Reactivity:
H M R

UniProt ID:
#P19484

Entrez-Gene Id:
#7942

CST Logo
Orders:

877-616-CELL (2355)

orders@cellsignal.com

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Color Storage Temp
TFEB Rabbit mAb Coated Microwells7826096 tests+4C
Phospho-TFEB (Ser211) Rabbit Detection mAb102811 eaRed (Lyophilized)+4C
HRP Diluent135155.5 mlRed+4C
TMB Substrate700411 ml+4C
STOP Solution700211 ml+4C
Sealing Tape545032 ea+4C
ELISA Wash Buffer (20X)980125 ml+4C
Cell Lysis Buffer (10X)980315 ml-20C

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.

Description

The rapid protocol (RP) PathScan® RP Phospho-TFEB (Ser211) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of TFEB protein phosphorylated at Ser211 in a reduced assay time of 1.5 hours. Incubation of cell lysates and detection antibody on the coated microwell plate forms a sandwich with phospho-TFEB (Ser211) in a single step. The plate is then extensively washed and TMB reagent is added for signal development. The magnitude of absorbance for the developed color is proportional to the quantity of phospho-TFEB (Ser211). Learn more about your ELISA kit options here.

*Antibodies in this kit are custom formulations specific to kit.

Specificity/Sensitivity

The PathScan® RP Phospho-TFEB (Ser211) Sandwich ELISA Kit detects endogenous levels of TFEB protein phosphorylated at Ser211. The kit sensitivity is shown in Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Background

Transcription factor EB (TFEB) is a member of the Myc-related, bHLH leucine-zipper family of transcription factors that drives the expression of a network of genes known as the Coordinated Lysosomal Expression and Regulation (CLEAR) network (1,2). TFEB specifically recognizes and binds regulatory sequences within the CLEAR box (GTCACGTGAC) of lysosomal and autophagy genes, resulting in the upregulated expression of genes involved in lysosome biogenesis and function, and regulation of autophagy (1,2). TFEB is activated in response to nutrient deprivation, stimulating translocation to the nucleus where it forms homo- or heterooligomers with other members of the microphthalmia transcription factor (MiTF) subfamily and resulting in upregulation of autophagosomes and lysosomes (3-5). Recently, it has been shown that TFEB is a component of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which regulates the phosphorylation and nuclear translocation of TFEB in response to cellular starvation and stress (6-9). During normal growth conditions, TFEB is phosphorylated at Ser211 in an mTORC1-dependent manner. Phosphorylation promotes association of TFEB with 14-3-3 family proteins and retention in the cytosol. Inhibition of mTORC1 results in a loss of TFEB phosphorylation, dissociation of the TFEB/14-3-3 complex, and rapid transport of TFEB to the nucleus where it increases transcription of CLEAR and autophagy genes (10). TFEB has also been shown to be activated in a nutrient-dependent manner by p42 MAP kinase (Erk2). TFEB is phosphorylated at Ser142 by Erk2 in response to nutrient deprivation, resulting in nuclear localization and activation, and indicating that pathways other than mTOR contribute to nutrient sensing via TFEB (3).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a registered trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) orders@cellsignal.com Support: 877-678-TECH (8324) info@cellsignal.com Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 2

Cell Signaling Technology Logo
Figure 1. Treatment of Raji cells with Torin 1 reduces phosphorylation of TFEB at Ser211, but does not affect the level of total TFEB. The relationship between lysate protein concentration from untreated and Torin 1-treated Raji cells and the absorbance at 450 nm using the PathScan® RP Phospho-TFEB (Ser211) Sandwich ELISA Kit #57002 is shown in the upper figure. The corresponding western blots using phospho-TFEB (Ser211) antibody (left panel) and TFEB antibody (right panel) are shown in the lower figure. Raji cells were either left untreated or treated with Torin 1 (250 nM) for 6 hr and then lysed.
ELISA Image 1: PathScan<sup>®</sup> RP Phospho-TFEB (Ser211) Sandwich ELISA Kit
Figure 2. TFEB is phosphorylated at Ser211 in untreated HeLa cell extracts, but is not phosphorylated in cell extracts treated with calf intestinal phosphatase (CIP) and λ phosphatase. The absorbance readings at 450 nm for HeLa cell extracts (400 μg/mL for each sample) that were either left untreated or treated with CIP/λ phosphatase using the PathScan® RP Phospho-TFEB (Ser211) Sandwich ELISA Kit #57002 are shown in the upper figure. The corresponding western blots using phospho-TFEB (Ser211) antibody (left panel) and TFEB antibody (right panel) are shown in the lower figure. HeLa cells were grown to 80% confluence, lysed, and then either left untreated or treated with CIP/λ phosphatase for 2 hr.
ELISA Image 2: PathScan<sup>®</sup> RP Phospho-TFEB (Ser211) Sandwich ELISA Kit
Orders: 877-616-CELL (2355) orders@cellsignal.com Support: 877-678-TECH (8324) info@cellsignal.com Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.