Biomedical Engineering Reference
In-Depth Information
Figure 3 . A prototypical strategy to isolate protein complexes. Letters A - E indicate proteins.
The bait protein A is reversibly bound through a chemical tag to a solid substrate that forms
part of an affinity chromatography column. Upon exposure to a protein extract from a cell,
proteins that bind to A, either directly or indirectly, are retained on the column. This complex
of proteins can be released from the column, and analyzed further. See text for details.
and that of protein A is generated. The fusion protein expressed from this gene
can be bound onto a solid support of glutathione-coated agarose beads that form
part of an affinity chromatography column. An important feature of this binding
is its reversibility, that is, the protein A-GST fusion (and anything else attached
to it) can be eluted from the solid support using an excess of glutathione.
When the chromatography column is exposed to a protein extract from a
cell, all proteins capable of binding (directly or indirectly) to protein A will be
retained on the column. After eluting protein A and its attached proteins, the
components of the protein complex are separated through gel electrophoresis.
Individual proteins in the complex can then be identified through mass spec-
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