an-electrochemiluminescence-based-assay-for-mecp2-protein-variants

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

Medical University of Vienna (MUV)

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease affecting 1 in 50,000 people and is caused by a GAA-trinucleotide expansion in the frataxin gene located on chromosome locus 9q13 which results in a markedly reduced expression of frataxin, a small mitochondrial protein. The exact function of frataxin is still unknown and currently there is no approved treatment available. In the near future there will be a high demand for measuring frataxin protein levels due to the development of therapeutic strategies for FRDA based on manipulating frataxin expression levels in vivo. In this paper we describe the development of an electrochemiluminescence assay (ECLIA) to measure frataxin protein levels in a 96-well plate format. The ECLIA for frataxin is able to measure human and mouse samples and is highly quantitative, accurate and reproducible, with low intra- and inter-assay error throughout a wide working range. The assay has an excellent precision and provides a new tool for the set up of high-throughput screening for basic research and for clinical studies with FRDA patients.

A high throughput electrochemiluminescence assay for the quantification of frataxin protein levels.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

Hannes Steinkellner

Institute of Medical Genetics,
Center for Pathobiochemistry and Genetics,
Institute of Medical Genetics, Center for Pathobiochemistry and Genetics

Medical University of Vienna (MUV)

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

Hannes Steinkellner

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Medical Genetics,Center for Pathobiochemistry and Genetics,Institute of Medical Genetics, Center for Pathobiochemistry and Genetics

Medical University of Vienna (MUV)

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

Institute of Medical Genetics,
Center for Pathobiochemistry and Genetics,
Institute of Medical Genetics, Center for Pathobiochemistry and Genetics