Immunoblot Analysis of Apoptosis Inhibition in Host Cells Expressing an Antiapoptotic Bacterial Protein

Take an SDS-PAGE gel containing resolved proteins from lysates of cells expressing CaeB, an anti-apoptotic bacterial protein, and from control cells lacking CaeB.

Both cell types carry an antibiotic-inducible expression system and were treated with the antibiotic to induce expression of a pro-apoptotic protein.

In control cells, this triggers apoptotic signaling, activating an apoptotic marker.

In CaeB-expressing cells, CaeB inhibits the apoptotic signaling, reducing apoptotic marker activation.

Using an electric field, transfer proteins from the gel onto a membrane.

Add a blocking solution to reduce non-specific antibody binding.

Incubate with a primary antibody targeting the activated apoptotic marker.

Wash to remove unbound antibody.

Incubate with an enzyme-conjugated secondary antibody targeting the primary antibody. Wash again, and add a chemiluminescent substrate that reacts with the enzyme to emit light.

Image the membrane.

A lower signal in CaeB-expressing cells compared to control cells suggests CaeB inhibits apoptosis downstream of the pro-apoptotic protein.

Blot the proteins onto PVDF membranes at a constant voltage of 16 volts for 60 minutes using a trans blot SD semi-dry transfer