Duplicate sets of sample preparation robotics are utilsed at the IFR and JIC. These include robotics for:

* 2-D gel electrophoresis

* gel staining/destaining

* gel imaging

* spot-picking

* proteolytic digestion

* MALDI-plate loading.

Descriptions of these instruments are given below:

Perkin-Elmer Arthur - Multi-Wavelength Fluorimager

Arthur is a multi-wavelength fluoro-imager capable of optical density and fluorescence measurements on a variety of media including 2D gels, microscope slides, tissue culture plates and exposed film. It has a maximum scanning area of 234 x 275 mm at resolutions of up to 50 um

Fluoroscence measurements can be made using combinations of up to excitation and emission wavelengths in the range 400 to 700 nm.

The sample is placed horizontally on the carrier plate (see photograph), which is automatically loaded into the instrument when a scan is requested. A cooled CCD camera scans the required area, capturing a number of FRAMES, which are combined to produce an IMAGE of the full sample.

A high resolution mosaic image is produced by illuminating the sample from:

i) below - with an ultraviolet light source for densitometry (transmission mode).

ii) above - with a high power xenon light source and interference filters for densitometry (reflection mode).

iii) above - with a high power xenon light source and interference filters for fluorescence measurements (top excitation mode).

iv) edge - with a high power xenon light source and interference filters for fluorescence measurements (translution mode).

iii) none - for chemiluminescence applications.

Investigator Flexys - Protein Picking Workstation.

The Investigator Flexys Protein Picking Workstation (photograph left) is a sophisticated application, specifically configured for the accurate and precise excision of protein spots from 2-D gels.

The major components of the Flexys are:

i) Dilutor unit
ii) Three-axis picking robot with gel imaging CCD camera and picking tool.
iii) Work area with: gel plate carrier, tip rack, output-plate rack, sonic bath, tip-wash station

The picking of protein spots is carried out by one of two methods: the Direct Method or the Blind Method.

The Direct Method - is used with gels that have a visible stain such as Coomassie Blue or Silver. The gel is placed on the workstation gel plate and imaged using the CCD camera mounted on the robot. The camera, connected to a PC, takes 42 separate pictures of the gel that are then built into a composite image on the PC. The image is analsed by the Bioimage software.

The Blind Method - is used for fluorescent-stained gels. The gel must first be imaged by a fluorescent- capable imaging device (see the Perkin-Elmer Arthur multi-wavelength fluorimager above) The resultant image is stored in the Bioimage database.
The same gel is then imaged on the Flexys using the method employed for imaging visibly-stained gels. The two images are then matched in the Bioimage software, using gel registration marks as anchor points. The software takes into account any relative shifts between the images and locates the protein spots.

From this point the procedure is the same for both gel types. It is possible to manually select the features of interest in the gel, or it is possible to link the image to other images in the gel database and use the advanced query features available in the Bioimage software.

Once the spots of interest have been identified and selected the Bioimage data is loaded into the Proteomics Picker software. The spots are then excised from the gel and transferred to output plates on the robot bed.

Investigator ProGest - Protein Digestion Station.

The Investigator ProGest Protein Digestion Station (left of photograph) is an automated instrument for the enzymatic digestion of proteins contained in pieces of gel after electrophoretic separation. The ProGest performs enzymatic digestion on up to 96 samples simultaneously.
It automates the following steps:

i) Washing of the gel slices with various buffers
ii) Reduction of disulfide bonds at 60 C
iii) Alkylation of cystine residues with iodoacetamide
iv) Removal of excess reagents
v) Equilibration with digest buffer
vi) Addition of digestion enzyme (optional up to 4 enzymes)
vii) Incubation at 37 C for digestion
viii) Elution of peptides from the gel matrix.

The major components of the system are:

i) Pipetting robot
ii) Dual syringe dilutor unit
iii) Work area with heatable reaction block, solvents and reagant rack, collection rack
iv) Built-in Touchpad and Liquid Crystal Display, single board computer, plus 3.5" disk drive and operating software
v) Protective cover.

Enzymatic digestion is a standard technique used as part of the sample preparation process for a number of analytical techiques which can include peptide mapping by liquid chromatography (LC) and capillary electrophoresis (CE), but most commonly by MALDI-ToF or MS/MS mass spectrometry. The profile of the peptide fragments generated by trypsin digestion (the most commonly used enzyme), can be considered to be a fingerprint for each protein. The masses of each peptide fragment from a protein 'fingerprint' can be accurately determined by mass spectrometry and these fragment sizes can be used to search against a variety of databases for a 'match'. From this 'match' the identity of the protein may be determined.

The MALDI-AutoPrep (MAP II) Robot.

The MALDI-AutoPrep (MAP II) Robot (right of above photograph) enables automatic preparation of batches of samples from solution vials onto the MALDI sample target. Features of the MALDI-AutoPrep (MAP II) Robot include:

i) protocols available for sample spotting using 'dried droplet', 'thin layer' or 'ZipTip' techniques.
ii) desalts samples on the target plate
iii) automatically sends spot co-ordinates and sample list to the Bruker Reflex III MALDI-ToF instrument.