Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells
Transcript
Begin with virus-infected mammalian cells that produce multiple caspases, including caspase-3.
Add a growth medium to the control well and a caspase-3 inhibitor-supplemented medium to the test well.
In the control well, caspase-3 recognizes and degrades the viral nucleoproteins and host proteins essential for viral propagation.
In the test well, the inhibitor enters the cell, inhibiting caspase-3 activity and preventing protein degradation.
Dislodge cells from the plate. Add a lysing reagent and heat to induce cell lysis, releasing intracellular proteins.
Load both samples on an SDS-polyacrylamide gel and electrophorese to separate proteins based on size.
After electrophoresis, transfer the gel to a blotting membrane.
Treat the membrane with primary antibodies against viral nucleoproteins and host proteins.
Wash and overlay with fluorophore-conjugated secondary antibodies binding with primary antibodies, generating fluorescent bands.
The higher fluorescence in the test sample indicates reduced caspase-3 mediated viral and host protein cleavage compared to the control.
