The discovery that bacterial plasmid vectors may be used for the synthesis of proteins which are “fused” together has resulted in the creation of a new class of proteins known as “fusion” proteins. The fused proteins, one of which has a strong affinity for a known ligand, may be easily purified using affinity chromatography. Examples of these fusion proteins are the Glutathione-S-Transferase (GST) fusion proteins, Maltose Binding Protein (MBP) fusion proteins and poly-histidine tagged fusion proteins.

Purification of GST fusion proteins using glutathione-agarose beads is well documented. Prior to purification, it may be useful to screen for the presence of GST proteins in cell lysates. We have immobilized glutathione through its central sulfhydryl to the wells of these plates. These glutathione coated plates can be used in the analysis of cell lysates to determine the presence and concentration of the GST-fusion protein. Because the plates are pre-blocked, initial purification of the cell lysates is not necessary. Specific binding of the protein is achieved through the interaction of GST with glutathione. Immobilization of GST fusion proteins using the glutathione coated microwell plates may also prove useful in the screening of sera for antibodies to the GST-fused protein.

Figure 1. Binding of GST to Pierce Glutathione Coated Plate.

Highlights:

• The lower detection limit is 1 ng of GST fusion protein
• Simple format for protein:protein interaction studies
• Detergents used to lyse cells do not inhibit protein binding to pre-coated plates as they do with plain polystyrene
• Pre-blocked with BSA and SuperBlock Blocking Buffer to reduce nonspecific binding
• Pre-blocked to reduce nonspecific binding
• 96-well plate in an 8-well strip plate format to maximize usefulness of plates
• Selectively binds recombinantly-expressed fusion proteins containing glutathione
  S-transferase

References:

1. Pullen, S.S., et al. (1999). J. Biol. Chem. 274, 14246-14254.
2. Yarwood, S.J., et al. (1999). J. Biol. Chem. 274, 14909-14917.
3. McKevitt, M., et al. (2003). Genome Res. 13, 1665-1674.

4. Pullen, S.S., et al. (1999). J. Biol. Chem. 274, 14246-14254.