Detection of GST-tagged Proteins with SDS-PAGE

Extracted from GST Gene Fusion System, GE Healthcare, 2014

SDS-PAGE is useful for monitoring tagged protein levels during expression and purification. Transformants expressing the desired tagged protein are identified by the absence of the parental GST and by the presence of a novel, larger tagged protein. Parental pGEX vectors produce a Mr 29 000 GST-tagged protein containing amino acids coded for by the pGEX multiple cloning site.

Reagents Required

2x sample loading buffer:

0.125 M Tris-HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 200 mM DTT. Store in aliquots at -20 °C.

DTT should be freshly prepared and added to the sample loading buffer just before adding the sample loading buffer to the samples. β-mercaptoethanol (500 µL per 10 mL) can be used as an alternative to DTT.

Gel Electrophorersis

1. Add 1 volume of 2x sample loading buffer to 1 volume of supernatant from crude extracts, cell lysates, or purified fractions, as appropriate.
   
   
2. Vortex briefly and heat for 5 min at 95 °C.
   
   
3. Centrifuge briefly, then load the samples onto an SDS-polyacrylamide gel.
   
   
4. Run the gel for the appropriate length of time and stain using preferred staining method.