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HTRF Human Total MDA5 Detection Kit, 500 Assay Points

The MDA5 kit is designed for the fast detection of total MDA5 in cell supernatants.

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).

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Feature	Specification
Application	Protein Quantification
Sample Volume	16 µL

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The MDA5 kit is designed for the fast detection of total MDA5 in cell supernatants.

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Product Variants

Unit Size: 500 Assay Points

Part #:

63ADK112PEG

List Price

USD 1,604.00

Unit Size: 10,000 Assay Points

Part #:

63ADK112PEH

List Price

USD 18,890.00

Product information

Overview

MDA-5 is an RIG-I-like receptor that acts as a cytoplasmic sensor of double stranded RNA, and plays a major role in sensing viral infection and in the activation of a cascade of antiviral responses including the induction of type I interferons and proinflammatory cytokines.

It is understood to form aggregates along RNA strands when performing its nucleic acid sensing functions.

It may also play an important role in enhancing natural killer cell function, and may be involved in growth inhibition and apoptosis in several tumor cell lines.

The MDA5 detection kit is compatible with our MDA5 aggregation kit, and enables the analysis of aggregated and total MDA5 from a single sample, for better a readout.

Specifications

Other Specifications

Application	Protein Quantification
Brand	HTRF
Detection Modality	HTRF
Molecular Modification	Total
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Biomarkers
Target Species	Human
Technology	TR-FRET
Therapeutic Area	Infectious Diseases Inflammation
Unit Size	500 Assay Points

Video gallery

How it works

MDA5 assay principle

The MDA5 assay quantifies the expression level of MDA5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The MDA5 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 MDA5 in a cell extract, the addition of these conjugates brings the donor fluorophore into 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 proteins expression under a no-wash assay format.

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MDA5 two-plate assay protocol

The 2-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of MDA5 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2biomarkers-how-it-works-assay-protocol-alpha-tubulin-63adk072peg-63adk072peh.svg

MDA5 one-plate assay protocol

Detection of MDA5 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.

2phospho-how-it-works-total-btk-1-plate-assay-protocol.svg

Assay validation

MDA5 aggregation in HUTU80 cells stimulated with poly I;C

HUTU80 cells were plated in 96-well plates in complete culture medium and incubated at 37°C, 5% CO2. The next day, cells were treated with Poly I:C (HMW)/LyoVec in low serum medium (0.5% FBS) for 2h at 37°C, 5% CO2. After this treatment, cells were then lysed with supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF aggregation MDA5 or Total-MDA5 detection reagents. The HTRF signal was recorded after ON incubation.

Simplified pathway

Function and regulation of MDA5

MDA5 acts as a cytoplasmic sensor of viral nucleic acids. Its ligands include mRNA lacking 2'-O-methylation at their 5' cap, and dsRNA over 1 kb long. When binding dsRNA, MDA5’s folding state is altered and triggers its aggregation. Aggregated MDA5 then goes on to bind MAVS, promoting IKK-related kinases: TBK1 and IKBKE. They in turn phosphorylate the interferon regulatory factors IRF3 and IRF7, which then activate transcription of antiviral immunological genes, including he type 1 interferons (IFNs) IFN-alpha and IFN-beta.