PlatformsneuroGENOMICS & neuroPROTEOMICS

   

2-dimensional gel electrophoresis A powerful tool for the separation of complex mixtures of proteins in two dimensions, first according to their net charge by an isoelectric focusing step and then, in the second dimension, according to their molecular weight employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) affording high separation efficiency. Separated proteins are then visualised by sensitive staining methods and identification following proteolytic digestion and mass spectrometric analysis. Proteome profile analysis also allows the comparison of protein expression between two samples, inherent limitations of electrophoresis overcome when coupled with techniques such as DIGE or ICAT.


Protein expression involves the cloning of a gene of interest into an expression vector, and highly expressing the protein in cellular systems. Following confirmation of successful transformation and protein expression, large quantities of experimental protein can be purified..




Mass spectrometry (MALDI-TOF) is a sensitive tool for the analysis of proteins by accurate measurement of the molecular masses of either intact protein or peptide fragments of proteolytically digested proteins allowing identification of unknown proteins after consultation with a specialised proteome database.

Chromatography - high resolution liquid chromatography (HPLC) is used for the isolation, identification and quantification of compounds by separation of complex mixes by passage through a column. The facility also has capabilities for capillary electrophoresis (HPCE) and capillary electrochromatography (HPCEC). Established preparative and analytical methods include the detection of serotonin and dopamine.

DNA microarrays are a powerful tool for global gene expression analyses, and the Platform offers automated microarray printing facilities, access to fluorescent sample labelling, hybridisation and scanning / signal measurement, and the skills of bioinformatics personnel adept at organising and mining microarray data output.
Gene expression analyses on the Affymetrix platform are provided in association with The Australian Genome Research Facility (www.agrf.org.au), bundled with the consultation services of our in-house bioinformatics services.

In situ hybridisation of paraffinised whole sections or tissue arrays extends microarray capabilities by enabling confirmation of identified expression patterns by histological analysis.

Restriction Fragment Length Polymorphism (RFLP) is a cost-effective method of high-throughput mutation / polymorphism screening, based upon the specific digestion of PCR-amplified DNA with restriction enzymes, and the differential separation of wild-type / anomalous sequences through polyacrylamide gel electrophoresis.

Serial Analysis of Gene Expression (SAGE) provides deep insight into global expression, involving the quantification of relative gene transcription using sequence tags unique to individual genetic transcripts.


Denaturing High Performance Liquid Chromatography (DHPLC) identifies mutations and polymorphisms due to melting temperature differences upon heteroduplex formation between mismatched nucleotides in double-stranded, PCR-amplified DNA. DHPLC is a sensitive, cost-effective method for high-throughput mutation detection.

Genetic diagnostics for neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Tauopathy, ALS and Creutzfeldt-Jakob Disease is offered by the Platform, in addition to the determination of loss of heterozygosity in brain tumours.

DNA sequencing & genotyping is performed with an ABI 3100 capillary electrophoresis system. Full service sequencing and technical assistance is available.

Image from Platform-generated data


Protein array technology is a developmental focus for the P&G Platform, with the aim of creating a valuable neuroscientific resource - a protein chip for mining global expression information of neurological proteins.
Current proteome profiling technologies have limited sensitivity in detecting and quantifying low-abundance proteins; whereas protein and peptide arrays are valuable tools for complementing transcriptional data (cDNA microarrays) in high-throughput, exquisitely sensitive expression analysis.
Protein chip development is a challenging process, demanding optimisation of antibody/protein production, capture reagents and cross-reactivity, and mastery of array generation - which will require the collaborative expertise of several Platform participants. The processing and analysis of commerically-available protein chips is currently available through the Platform, including experimental desgin, protein labelling and hybridisation, scanning and data analysis.

Neuroscientific mutation database development is a key aim of the Platform, and will provide a valuable resource to the international scientific and clinical communities. Biological information (including expression patterns, mutations, clinical data and research links) of a broad array of neurological genes will be organised into a web-accessible database to be maintained through the Platform and collaborating international bodies.

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