2D ElectrophoresisThe Aberdeen Proteome Facility provides facilities and expertise for a range of analytical methods. Interested users of the Proteome Facility should contact Dr. Phil Cash (email: p.cash@abdn.ac.uk) to discuss their requirements in detail.
Aberdeen Proteome Faciity Laboratories.
Conditions of service (summary)
The Aberdeen Proteome Facility (APF) is fully equipped for the analysis of protein samples using both small- and large-format 2-Dimensional Gel Electrophoresis (2DGE). The large format gels use standard commercial systems, specifically the DALT (Amersham-Pharmacia) and Investigator (Genomic Solutions) equipment. Immobilised pH gradient gel electrophoresis is used throughout for the first dimension gel electrophoresis, although carrier ampholyte based separations can be used on request. Batch processing of the samples by 2DGE maintains gel-to-gel reproducibility. The current capacity of the Facility is up to 30 small- (run as batches of 10 gels) or 20 large-format DALT gels (run as batches of 10 or 20 gels) processed per week. All standard detection methods (Coomassie Brilliant blue, silver and fluorescent stains) as well as immunoblotting can be used.
Gel profiles are digitised using either CCD cameras or laser densitometry and computer analysis of the gel profiles uses the latest version of 'Phoretix 2D' analytical software.
Advice will be provided in advance for optimal sample preparation and any pre-selection processes required for specific protein classes. If required we will carry out optimisation for sample preparation in collaboration with users.
Selection of 2D gel format and protein detection.
Aberdeen-based researchers can download a copy of our record form used when submitting samples for analysis by 2DE. The completed form can be brought to the lab with your samples. Copies of the submission form can also be collected from the 2D gel lab.
A number of analytical systems are available for the identification of proteins extracted from gels. Samples are processed either from gels handled at the APF or sent directly by users. For user-supplied samples additional "cleaning" of samples can be carried out at the APF using 1-Dimensional electrophoresis and HPLC.
Mass Spectrometry and Peptide Mass Mapping
Peptide mass mapping is routinely used for the post-electrophoretic identification of proteins extracted from 1D
and 2D electrophoresis gels. The APF is fully equipped for automated processing of samples for peptide mass mapping
and uses the Genomic Solutions robots for spot cutting from gels, sample digestion, peptide extraction and sample
spotting for mass spectrometry. This equipment minimises keratin contamination of the samples. Peptide profiles
are determined using an ABI Voyager DE-STR MALDI-TOF instrument to achieve mass accuracies of 10-100 ppm for individual
peptides. The current capacity of the Facility for peptide mass mapping is 10-20 samples per day providing a full
database search report for protein identification.
Intact protein mass spectra can also be determined with a mass accuracy of 1000 ppm for proteins of masses up to 300 kDa.
EDMAN sequencing
N-terminal and internal amino acid sequences of proteins transferred
to membranes are determined by automated Edman sequencing. The APF is equipped with 2 ABI protein sequencers. The
current capacity of the sequencing facility is 5-10 samples per week. Sequences of 5-30 amino acids can be achieved
from 0.5-20 pmoles of protein, dependent upon the sample quality.
Database searching can be performed on the sequences obtained for protein identification.
