Automated 2D gel digestion in constructing a proteome map for by 0y3UmzEf

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									Automated 2D gel digestion in constructing a proteome map of
Campylobacter jejuni

    Antony Jones
    The Functional Genomics Laboratory
    School of Biosciences
    The University of Birmingham
    Edgbaston
    Birmingham B15 2TT
    United Kingdom
    Email : a.c.jones.bcs@bham.ac.uk

Overview
-     Whole cell protein extracts from Campylobacter jejuni were resolved using 2D gel analysis
-     Gels were visualised using coomassie staining, and spots of interest were excised from gels
-     Gel plugs in 96 well microtitre plates were destained, and proteins contained therein were trypsinised
      and extracted from the plugs using a fully-automated procedure on the RoboSeq4204


Introduction
The Functional Genomics Laboratory at the University of Birmingham houses a state-of-the-art genomics &
proteomics facility. The automated biosystems from MWG Biotech that are central to this facility have greatly
assisted the daily operation of the laboratory through performing a range of genomics applications such as
plasmid DNA isolation and DNA sequencing reaction set-up. As interest in proteomics continues to increase
rapidly, the need has arisen for effective automated solutions for related applications. Here we describe a
well-established method for automated 2D gel digestion. As the method uses simple ‘off the shelf’ reagents,
it is also highly cost-effective in comparison with the kit-based technologies currently available.




                                                                            MWG Biotech’s RoboSeq4204 can be
                                                                            custom-configured across a range of
                                                                            applications. The user can switch
                                                                            between configurations in seconds.
Method
1.   Gel plugs were destained by sequential washing with acetronitrile and ammonium bicarbonate
2.   Proteins were then stabilised by consecutive treatment with dithiothreitol and iodocetamide
3.   Treatment with trypsin restricted proteins into peptide fragments of a suitable size for MS analysis
4.   Peptides were released from the gel plugs by the addition of 1% formic acid & 2% acetonitrile
5.   Extracted peptides were thereafter loaded directly onto the MALDI-TOF MS instrument


Results




The figure shows a typical MALDI-TOF trace obtained from samples extracted on the RoboSeq4204. Data
obtained were used in constructing a proteome map for Campylobacter jejuni

								
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