Instrumentation

Electrophoresis has been a valuable method of protein analysis for decades. In recent years, the increased sensitivity of automated sequencers and analyzers has enabled the use of electrophoretic separation techniques to be used as preparative methods for subsequent stages of high-sensitivity protein analysis. The Laboratory is fully equipped to perform a variety of routine electrophoretic separations of proteins in polyacrylamide or agarose matrices. Sodium dodecyl sulfate polyacrylamide (SDS) and non-denaturing gels are available in standard and mini-gel format (linear and gradient format) as are 2-dimensional electrophoretic separations. Analytical isoelectric focusing (IEF) is performed on an Amersham Pharmacia Biotec IPGPhor electrophoresis module (first dimension) using immobilized pH gradients in polyacrylamide strips (DryStrips™). The second dimension of resolution is obtained on a vertical slab gel electrophoresis unit equipped with an external re-circulating cooling device.

Electroblotting of proteins to solid membrane supports has been used for many years in protocols used for immunodetection of antigens. Recent developments in membrane support technology have led to the production of high-capacity, chemically inert matrices that are resistant to N-terminal sequencing chemistries. Proteins blotted to these matrices can be excised from a mixture of proteins and subjected directly to sequence analysis or preliminary manipulations such as direct In situ chemical or proteolytic cleavage with subsequent peptide extraction and purification. The Laboratory is equipped to perform electroblotting procedures as well as In situ or "in gel" digestion procedures as required to obtain sequence information and protein identity.