Description
Formats:
Our HV (100 assay point) kits allow you to run 100 wells in 96-well format, using a 100 µL reaction volume (10 µL of sample).
Our 500 assay point kit allows you to run 500 wells in 96-well or 384-well format, using a 50 µL reaction volume (5 µL of sample).
Our 5,000 assay point kit allows you to run 5,000 wells in 96-well or 384-well format, using a 50 µL reaction volume (5 µL of sample).
Features:
No-wash steps, no separation steps
ELISA alternative technology
Sensitive detection
Broad sample compatibility
Small sample volume
Results in less than 3 hours
Half the time of an ELISA assay
Frataxin is expressed as a 210 AA, 23 kDA protein from the FXN gene located on chromosome 9. Upon expression, the FXN protein is directed to the mitochondrion by its 41 AA N-terminal mitochondrial targeting sequence. In the mitochondrion, the protein is cleaved by the mitochondrial processing peptidase (MPP) to its intermediate form of 42 – 210 AA. Later on, MPP cleaves the protein to its mature form of 81-210 AA. Although the function of the FXN protein is not clearly defined, it is thought to be vitally important for Iron-Sulfur cluster biogenesis, heme biosynthesis, and chelation and transportation of iron specifically involved with the mitochondria. The Frataxin protein is the primary culprit for a debilitating neurodegenerative disease called Friedreich’s Ataxia. Due to similarities in the diseases, there is some evidence to suggest that FXN may be involved in other neurodegenerative diseases such as Parkinson’s, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis. This AlphaLISA kit has been designed for the detection and quantification of FXN from cell and tissue lysates.
AlphaLISA technology allows the detection of molecules of interest in a no-wash, highly sensitive, quantitative assay. In an AlphaLISA assay, a biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads causes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.
Principle of the AlphaLISA assay
The AlphaLISA assay is based on an AlphaLISA sandwich immunoassay involving a biotinylated anti-analyte antibody bound to Streptavidin-coated AlphaLISA Donor beads and an anti-analyte antibody conjugated to AlphaLISA Acceptor beads. Both antibodies are directed against the analyte of interest. In the presence of the target, both antibodies bind to analyte and the beads come into proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer within the Acceptor beads, resulting in emission with λmax at 615 nm. The intensity of the signal is directly proportional to the concentration of analyte present in the sample.
Protocol of the AlphaLISA assay
The AlphaLISA assay can be run in a 96- or 384-well detection plate (50 µL final). As described here, samples or standards are dispensed directly into the assay plate for the detection of the analyte of interest by AlphaLISA reagents. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.
