Detection of GST-tagged Proteins with Western Blot

Expression and purification of GST-tagged proteins can be monitored by Western blot analysis, using Amersham ECL, Amersham ECL Prime, or Amersham ECL Select detection systems to enhance sensitivity.

Table 1. Reagents Required

Anti-GST antibody (goat polyclonal)
Blocking/incubation buffer:	5% (w/v) nonfat dry milk and 0.1% (v/v) Tween 20 in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3)
Wash buffer:	0.1% (v/v) Tween 20 in PBS (as above), referred to as PBS-Tween in the protocol below OR 0.1% (v/v) Tween 20 in 1x TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4 to 8), referred to as TBS-Tween in the protocol below

Secondary antibody to detect the anti-GST antibody (such as anti-goat IgG HRP conjugate)

Appropriate membrane, such as Amersham Hybond™ ECL (nitrocellulose) or Amersham Hybond-P (PVDF)

Electrophoretic Separation of Proteins

1. Separate the protein samples by SDS-PAGE.

Although anti-GST antibody from Cytiva has been cross-adsorbed with E. coli proteins, low levels of cross-reacting antibodies may remain. Samples of E. coli lysates that do not contain a recombinant pGEX plasmid and samples that contain the parental pGEX plasmid should always be run as controls.

1. Transfer the separated proteins from the electrophoresis gel to the membrane.

Electrophoresis and protein transfer can be accomplished using a variety of equipment and reagents.

Blocking of Membrane

1. Transfer the membrane onto which the proteins have been blotted into an appropriately sized container, such as a Petri dish.
2. Add 50 to 200 mL of blocking/incubation buffer to the container.
3. Incubate with agitation for 1 h at room temperature, or at 37 °C if the background is persistently and unacceptably high. Alternatively, membranes may be left in the blocking solution overnight at 2 °C to 8 °C, if more convenient.
4. Decant and discard the buffer.
5. Briefly rinse the membrane in wash buffer.

Longer incubation times with blocking/incubation buffer may reduce background signal.