title: Evaluation of protein loading techniques and improved separation in OFFGEL isoelectric focusing.
abstract: 2-DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off-gel) of proteins with the help of a 12- or 24-well frame. With this instrument also conventional focusing in IPG strips after passive in-tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup-loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in-tray rehydration loading. Especially, the recovery of basic and high-molecular-mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48-well OFFGEL frame was developed, which doubles the resolution of the commercially available 24-well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.
authors.affiliation: Max-Planck-Institute of Biochemistry, Protein Analysis Group, Martinsried, Germany. keidel@biochem.mpg.de
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Keidel
authors.forename: Dominik
authors.initials: D
authors.lastname: Dosch
authors.forename: Achim
authors.initials: A
authors.lastname: Brunner
authors.forename: Josef
authors.initials: J
authors.lastname: Kellermann
authors.forename: Friedrich
authors.initials: F
authors.lastname: Lottspeich
mesh_terms: D015180:Electrophoresis, Gel, Two-Dimensional; D004867:Equipment Design; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification*; D007091:Image Processing, Computer-Assisted; D007525:Isoelectric Focusing / Q000295:instrumentation* / Q000379:methods*; D007526:Isoelectric Point; D025341:Principal Component Analysis; D020033:Protein Isoforms; D040901:Proteomics / Q000379:methods*; D018892:Proton-Motive Force; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D029968:Escherichia coli Proteins; D020033:Protein Isoforms