Protein Name & Synonyms:
ETS domain-containing protein Elk-1
- IGF Signaling
- MAPK Signaling
- PI3K-AKT Signaling
The olionucleotide/antibody pair provided in this kit recognizes human ELK-1 in whole lysates and nuclear extracts.
Number of Targets Detected:
Compatible Sample Types:
- Cell Lysates
- Nuclear Extracts
Method of Detection:
1, 2, or 5 x 96-Well Strip Microplate Kit
MAP kinase (MAPK) pathways including ERK1/2, p38 and JNK are activated upon environmental stimulus by growth factors or cellular stress, such as UV irradiation. This signaling serves to transmit external stimuli to the nucleus and activate numerous transcription factors. This results in changes in gene expression required for cellular growth, differentiation and survival. One downstream effector of these signaling pathways is Elk-1, a transcription factor that regulates immediate early gene (IEG) expression via the serum response element (SRE) DNA consensus site. Elk-1 belongs to the ternary complex factor (TCF) subfamily, part of Ets (E twenty-six) oncogene family of transcription factors which contain a highly conserved DNA-binding domain (DBD, the Ets domain) comprised of 85 amino acids. The TCF subfamily consists of three members ELK-1, SAP-1 and SAP-2/Net which are distinct gene products with different chromosomal localizations in both the mouse and human genomes. Each of the TCFs contains three conserved domains including the N-terminal Ets DNA binding domain, B-box region engaged in the interaction with serum response factor (SRF) to form ternary complex, and C-terminal transcriptional activation domain. Elk-1 can be activated by either pathway of ERK, JNK, or p38 through phosphorylation and de-SUMOylation. SUMO modification of Elk-1 by ERK pathway represses its transcriptional activity via SUMO dependent recruitment of histone deacetylases. Phosphorylated and de-sumoylated ELK-1 translocates to the nucleus from the cytoplasm and binds to SRE DNA sequences containing a central GGAA/T motif to initiate expressions of involved genes.
- 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
- 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
- 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
- Microplate reader capable of measuring absorbance at 450 nm
- 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.
Transcription factor assay of ELK-1 from nuclear extracts of HeLa cells or HeLa cells treated with PMA for 3 hr. After stimulation activated ELK-1 binds with its corresponding DNA with the RayBio® TF Activity Assay Kit.
Transcription factor assay of ELK-1 from nuclear extracts of HeLa cells or HeLa cells treated with PMA for 3 hr with the specific competitor or non-specific competitor. The result shows specific binding of ELK-1 to the ARE binding site detected by using the RayBio® TF Activity Assay Kit.
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.