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2D Gels
By Andrew Garrow,
University of Leeds
Myself
PhD – Bioinformatics for a proteomics
approach to understanding the
Schistosome tegument.
Construction of a proteomics database
2D Gels
Mass spectrometry
Data analysis
Lecture Layout
Biological sample
Sample prep.
2D Gel separation
Imaging
Protein excision
Protein digestion
MS/Protein ID
Global bioinformatics
Proteomic discovery
2D Gels
Use narrow range IPG strips to focus on particular pI ranges.
2D Gel Procedure
•Coomassie Blue R-250 40ng 2.5hr/2 steps Oldest and least expensive method
•Silver Stain Plus 1ng 1.5hr/3 steps MS compatible; high sensitivity; low
background
•Bio-Rad silver stain 1ng 2hr/7 steps High sensitivity; detects some highly
glycosylated and other difficult to
stain proteins
Software features
Spot detection
Spot quantification
Noise reduction
Gel comparison by warping
Linkage with robots
Robot Spot Cutter
Post Gel analysis
www.expasy.ch - Swiss-2DPAGE
http://www.harefield.nthames.nhs.uk/nhli/protein/index.html - HSC-
2DPAGE, Heart Science Centre, Harefield Hospital
Rules:
Rule 1 – Individual entries in the database must be accessible by a keyword
search. Other methods are possible but not required.
Rule 2 – The database must be linked to other databases by active hypertext
cross-references, linking together all related databases. Database entries
must be at least linked to the main index.
Rule 3 – A main index has to be supplied that provides a means of querying
all databases through one unique query point. Currently, the main index is the
SWISS-PROT database.
Rule 4 – Individual protein entries must be available through clickable images.
Rule 5 – 2DE analysis software designed for use with federated databases,
must be able to access individual entries in any federated 2DE databases.
http://ca.expasy.org/ch2d/fed-rules.html
Swiss 2DPAGE
Established in 1993
Maintained by the Central Clinical Chemistry
Laboratory of the Geneva University Hospital
and the Swiss Institute of Bioinformatics.
Entries highly annotated -
containing textual data on proteins including:
mapping procedure
physiological and pathological information,
http://babbage.csc.ucm.es/2d/2d.html -
COMPLUYEAST 2DPAGE, Saccharomyces cerevisae 2D-PAGE database at
Universidad complutense Madrid, Spain
http://www.gram.au.dk/ -
PHCI-2DPAGE, Parasite host cell interaction 2D-PAGE interaction database.
http://www.bio-mol.unisi.it/2d/2d.html -
Sienna 2D PAGE
Appel RD, Bairoch A, Sanchez JC, Vargas JR, Golaz O, Pasquali C and Hochstrasser
DF, 1996 – Federated two-dimensional electrophoresis database: a simple means of
publishing two-dimensional electrophoresis data, Electrophoresis, 17, 540-546.