This HTRF kit enables the cell-based quantitative detection of total TDP43.
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).
TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein involved in RNA-related metabolism. Aggregated TDP-43 has been identified as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), and more widely in several neurodegenerative diseases: TDP-43 proteinopathies. In pathological conditions (mutation or dysregulation), TDP-43 forms insoluble inclusion bodies in the cytoplasm of neurons in the brain and spinal cord. The TDP-43 Phospho-Ser409/410 assay detects human TDP-43 phosphorylated in cell lysates.
Application |
Cell Signaling
|
---|---|
Brand |
HTRF
|
Detection Modality |
HTRF
|
Molecular Modification |
Total
|
Product Group |
Kit
|
Sample Volume |
16 µL
|
Shipping Conditions |
Shipped in Dry Ice
|
Target Class |
Phosphoproteins
|
Technology |
TR-FRET
|
Unit Size |
500 Assay Points
|
The HTRF Total TDP-43 assay quantifies the expression level of TDP-43 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total TDP-43 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 TDP-43 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.
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of the Total TDP-43 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Total TDP-43 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.
Neuro2a cells were cultured in a 96-well plate (50,000 cells/well) for 24h and then treated for 30 min with increasing concentrations of Calyculin A (protein phosphatase 1 and 2A inhibitor). After treatment, cells were lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30 min at room temperature under gentle shaking (as per the suspension cell protocol).
After lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-TDP-43 (Ser409/410) or Total TDP-43 detection antibodies were added. The HTRF signal was recorded after an overnight incubation at room temperature.
As expected, Calyculin A triggered a dose-dependent accumulation of phosphorylated TDP-43 at Ser409/410 through inhibition of PP1/2, while the expression level of the protein was not modulated by the treatment.
Neuro2a cells were cultured in a 96-well plate (50,000 cells/well) for 24h, and then treated for 1h30 with the Casein Kinase 1 (CK1) inhibitors IC 261 or PF 670462 (100 µM), followed by Calyculin A activation (30 min, 100 nM).
After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-TDP-43 (Ser409/410) or Total TDP-43 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.
As expected, the results obtained showed an inhibition of TDP-43 Ser409/410, triggered by Calyculin A after CK1 inhibitor treatment, while the expression level of the protein was not modulated by the treatment.
Adherent human & mouse cells Neuro 2A, HeLa, and SH-SY5Y were seeded at 50,000 cells/well in a 96-well microplate. After a 24h incubation, the cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.
Following lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Total TDP-43 detection reagents. The HTRF signal was recorded after an overnight incubation.
The HTRF Total TDP-43 assay efficiently detected total TDP-43 in various human and mouse cell models with different expression levels.
HeLa cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were treated with Calyculin A (30 min, 100 nM), then lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total TDP-43 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.
Using the HTRF Total TDP-43 assay, 1,000 cells/well were enough to detect a significant signal, while 4,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF Total TDP-43 assay was 4 times more sensitive than the Western Blot technique.
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