Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis.
A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.
Roncada
Paola
P
Istituto Sperimentale Italiano, L. Spallanzani, Milan, Italy.
Cretich
Marina
M
Fortin
Riccardo
R
Agosti
Susanna
S
De Franceschi
Lucia
L
Greppi
Gian Franco
GF
Turrini
Francesco
F
Carta
Franco
F
Turri
Stefano
S
Levi
Marinella
M
Chiari
Marcella
M
D000180:Acrylic Resins; D001798:Blood Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005782:Gels; D020543:Proteome / Q000032:analysis*; D012685:Sepharose / Q000031:analogs & derivatives; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000180:Acrylic Resins; D001798:Blood Proteins; D005782:Gels; D020543:Proteome; C505993:acrylamide-allyl agarose copolymer; D012685:Sepharose