ABSTRACT
Two-dimensional gel electrophoresis is the combination of two high-resolution electrophoretic procedures (isoelectric focusing and SDS-polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first-dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS-slab gel and electrophoresed. The proteins in the first-dimension gel migrate into the second-dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in 1975. For very basic or very acidic proteins, two alternate protocols are provided. A third alternate protocol describes how two-dimensional electrophoresis can be performed using a minigel system. Protein sample preparation is presented in the support protocol.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Gel, Two-Dimensional/instrumentation , Electrophoresis, Polyacrylamide Gel/instrumentation , Electrophoresis, Polyacrylamide Gel/methods , Hydrogen-Ion Concentration , Isoelectric Focusing/instrumentation , Isoelectric Focusing/methods , Proteins/chemistryABSTRACT
Two-dimensional gel electrophoresis is the combination of two high-resolution electrophoretic procedures (isoelectric focusing and SDS-polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first-dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS-slab gel and electrophoresed. The proteins in the first-dimension gel migrate into the second-dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in 1975. For very basic or very acidic proteins, two alternate protocols are provided. A third alternate protocol describes how two-dimensional electrophoresis can be performed using a minigel system. Protein sample preparation is presented in the support protocol.
Subject(s)
Electrophoresis, Gel, Two-Dimensional/methods , Proteins/analysis , Electrophoresis, Gel, Two-Dimensional/instrumentation , Electrophoresis, Polyacrylamide Gel/instrumentation , Electrophoresis, Polyacrylamide Gel/methods , Isoelectric Focusing/instrumentation , Isoelectric Focusing/methods , Isoelectric Point , Molecular WeightABSTRACT
Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.
