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Product Description
Specifications
| Size | 1 Plate Kit, 2 Plate Kit, 5 Plate Kit |
| Estimated Lead Time | 1-2 weeks |
| Species | Human |
| Specificity | The olionucleotide/antibody pair provided in this kit recognizes human TFEB in whole lysates and nuclear extracts. |
| Compatible Sample Types | Cell Lysates, Nuclear Extracts |
| Accession Number | P19484 |
| Gene Id | 7942 |
| Gene Symbols | TFEB, BHLHE35 |
| Protein Name / Synonyms | Transcription factor EB Class E basic helix-loop-helix protein 35 (bHLHe35) |
| Quantitative/Semi-Quantitative | Semi-Quantitative |
| Solid Support | 96-well Microplate |
| Design Principle | Sandwich-based |
| Method Of Detection | Colorimetric |
| Research Area | Autophagy |
| Shipping Type | Dry ice (additional shipping fee) |
| Storage | -20°C |
Introduction
To respond to environmental changes of cellular nutrient insufficiency, cells trigger autophagy cooperating autophagosomes and lysosomes to maintain energy homeostasis. Dysfunction of this cellular catabolic process involves serious diseases such as liver disease and neurodegeneration diseases. The transcription factor EB (TFEB) is a master gene for lysosomal biogenesis. It regulates expression of over 100 autophagy and lysosomal genes. TFEB has basic helix-loop-helix and leucine zipper domains, which places it in the micropthalmia-transcription factor E (MiT/TFE) subfamily along with TFE3, TFEC and MITF. In normal situations, TFEB is phosphorylated through pathways related with mTOR and MAPK and stays in cytoplasm. Upon stimulation of starvation of nutrients, the TFEB is activated by loss of phosphrylation and translocates into the nuclear where it binds to a CLEAR (Coordinated Lysosomal Expression and Regulation) consensus DNA sequence in the promoters of lysosomal genes to regulate expression of downstream genes.
Product Features
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
Application Notes
Kit Components
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- DTT
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- TMB One-Step Substrate Reagent
- Stop Solution
Other Materials Required
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation <lIκBenchtop rocker or shaker
- Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Typical Data
Figure 1
Transcription factor activity assay of TFEB from nuclear extracts of HepG2 cells or HepG2 cells treated with HBSS medium for 4 hr. After stimulation, activated TFEB is translocated into the nucleus where it binds with its corresponding DNA. A. Western-blot result of TFEB from cytoplasm and nuclear fractions. B. Transcription factor activity assay of TFEB from nuclear fractions with the RayBio® TFEB Transcription Factor-Activity Assay Kit.
Figure 2
Transcription factor activity assay of TFEB from nuclear extracts of HepG2 cells or HepG2 cells treated with HBSS medium for 4 hr with the specific competitor or non-specific competitor. The result shows specific binding of TFEB to the TFEB conserved binding site.
Storage/Stability
Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.
