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HTRF Human Total ADAR1 Detection Kit, 500 Assay Points
HTRF Human Total ADAR1 Detection Kit, 500 Assay Points
Shipping box for Revvity reagent kits
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Sample Volume | 16 µL |
Product variants
Unit Size: 500 assay points
Part #:
64ADAR1TPEG
Unit Size: 10,000 assay points
Part #:
64ADAR1TPEH
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Human Total ADAR1 Detection Kit, 500 Assay Points
Shipping box for Revvity reagent kits
HTRF Human Total ADAR1 Detection Kit, 500 Assay Points
Product information
Overview
ADAR1 is one of two ADAR gene-coded enzymes. It binds to double-stranded RNA (dsRNA) and deaminates its adenosine into inosine (hypoxanthine). ADAR proteins act post-transcriptionally to alter RNA nucleotide contents. The deamination they operate interferes with the usual A:U pairing and destabilizes the RNA.
The loss of regulation of ADAR1 & ADAR2 is involved in the development and progression of multiple cancers, such as glioblastomas, melanomas, or acute leukemias. On top of their role in oncogenesis, ADAR enzymes are suspected of contributing to the aggravation of some infectious diseases like HIV and measles, but also of mental disorders such as depression, epilepsy, and schizophrenia.
How it works
Total ATG16L1 assay principle
The HTRF Total-ATG16L1 assay quantifies the expression level of ATG16L1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total-ATG16L1 assay uses two labeled antibodies, one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of ATG16L1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total-ATG16L1 two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Total ADAR1 detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-ATG16L1 one-plate assay protocol
Detection of Total ADAR1 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Assay validation
Validation of Total ADAR1 detection in siRNA-treated HepG2 cells
HepG2 cells were plated at different cell densities under 80µl in a 96-well plate in complete culture medium, and incubated overnight at 37°C, 5% CO2. After culture, the cells were treated with siRNA by adding 20µl of a mix of Lipofectamine® RNAiMax/siRNA for ADAR1, then incubated for 24h and 48h at 37°C, 5% CO2. For untreated cells, 20µl of OptMEM medium were added instead of the siRNA mix. After incubation, cells were lysed with 50µL of supplemented 1X lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before adding 2µL of the HTRF d2 detection reagent and 2µL HTRF Eu-K detection reagent. The HTRF signal was recorded after an ON incubation.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Lysis Buffer Compatibility |
Lysis Buffer 1
Lysis Buffer 2
Lysis Buffer 3
|
| Molecular Modification |
Total
|
| Product Group |
Kit
|
| Sample Volume |
16 µL
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
TR-FRET
|
| Unit Size |
500 assay points
|
Video gallery
HTRF Human Total ADAR1 Detection Kit, 500 Assay Points
HTRF Human Total ADAR1 Detection Kit, 500 Assay Points
Resources
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