The Technique

Electrophoresis:  2D polyacrylamide gel electrophoresis (2D PAGE) allows protein separation using protein pI in the first dimension, and size in the second, potentially capable of separating thousands of proteins with high resolution.  The proteomics facility maintains two IPGphor units for first-dimension separation by isoelectric focusing as well as vertical electrophoresis units for molelular weight separation.

Imaging:  quantitative high-resolution imaging is a fundamental requirement for proteomic experiments and is broadly applicable for other investigations where quantification of labelled or stained gels is required. A Typhoon 9400 variable mode imager is employed for fluorescence imaging, and is also capable of performing phosphorescence and chemiluminescence detection. An infrared scanner (Odyssey) is also available for quantification of protein stains (eg, colloidal coomassie blue).  A transmittance scanner is also available for coomassie and silver stained gels and for x-ray films.

Applications

The principal technique involves running and comparison of 2D polyacrylamide gels, followed typically by mass spectrometry for identification of the protein spots cut from the gels.  The comparative nature of the technique allows identification of protein expression changes across differerent samples.  Differential In-Gel Electrophoresis (DIGE) is an extension of 2D PAGE in which proteins are fluorescently labelled with Cy-dyes prior to 2D separation, allowing greater resolution and reduced variability in sample analysis.

Facilities available

The facility is located in the Molecular Biosciences Building and includes equipment for proteomics, gel imagng and general protein sample preparation.

  1. Typhoon Variable Mode Scanner (Phospho-storage, chemiluminescence and fluorescence)
  2. IPGphor Iso-electric focussing units (7, 13 and 24 cm IEF strips)
  3. Ettan Dalt II 2nd dimension Electrophoresis System and Gel Caster (24 cm)
  4. Progenesis SameSpots software for single and multi-stained gels
  5. Waterbath Sonicator
  6. Thermo Savant: Speedvac and Freeze Dryer
  7. HP Photo Scanjet
  8. Odyssey Infra-Red Imager
  9. SpectraMAX 190 Plate Reader (U/V vis)
  10. Eppendorf refrigerated bench top microcentrifuge
  11. Umax Image scanner for transmittance scanning of film and gels.

Other equipment located within the Facility includes:

  1. Agilent Bioanalyser:  for RNA, DNA and protein spectral imaging
  2. Microcal iTC200 microcalimeter
  3. Biacore 3000
  4. Beckman Multimode detector
  5. Beckman Optima XL/I analytical ultracentrifuge
  6. AKTA fplc

Users should contact Dr Steven Mason for mor information regarding this equipment.

Sample requirements

Experimental approaches and sample requirements should be discussed with Dr Amanda Nouwens or Dr Steven Mason.  All reagents must be supplied by the researcher.

Analysis of data

Data analysis should be discussed with Dr. Amanda Nouwens or Dr Steven Mason.

Contacts

Dr Amanda Nouwens
Email: a.nouwens@uq.edu.au
Phone:  3346 9490

Dr Steven Mason
Email: steven.mason@uq.edu.au
Phone: 3365 4872

Charges for use

Other UQ: $2,000 per year or $200 per month

Outside UQ:  price on application