2-D Gel Electrophoresis
The technique of two-dimensional electrophoresis involves
separating proteins in the first dimension according to charge
(isoelectric focusing, IEF), followed by separating the focused
proteins in the second dimension according to molecular weight by
sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). The
proteins may be visualized by staining with Coomassie Brilliant Blue
R250, silver stain, fluorescent dyes or through radioisotope
detection (phosphorescence) after the proteins are metabolically
labeled with 3H, 14C, or 35S labeled amino acids. This
two-dimensional array will produce spots that correspond to a single
protein species in the sample. Using this technique combined with
the 2D gel analysis system, thousands of different protein can be
separated and information such as pI, molecular weight and protein
amount can be determined. These spots can be excised for further
analysis or the 2-D array can be analyzed for differences in protein
quantity or in proteins present in the gel. 2-D gels can also be
electroblotted to PVDF or nitrocellulose membranes for further
analysis.
2-D electrophoresis is performed using the IPGPhor system from
Amersham Pharmacia Biotech,
for the1st dimension separation, followed by 2nd dimension separation
using either a Bio-Rad
Mini-Protean II system or the Amersham
Pharmacia Biotech Hoefer DALT system (20 cm x 23 cm gels).
