Detection of Hepatitis C Virus Replication Protein in Transfected Cells by Western Blotting

Begin with a Hepatitis C virus RNA-transfected human hepatoma cell lysate containing the host protein beta-actin and viral proteins, including the NS3 viral replication protein.

Beta-actin acts as an internal control for protein expression.

Add a loading buffer and load the lysate onto an SDS-PAGE gel.

Initiate electrophoresis to separate the proteins by molecular weight, producing discrete protein bands.

Transfer the proteins from the gel onto a membrane.

Treat the membrane with a blocking solution to reduce non-specific antibody binding, then wash.

Incubate the membrane with a mixture of primary monoclonal antibodies specific to NS3 and beta-actin.

The antibodies bind to their target proteins, then wash.

Add an enzyme-conjugated secondary antibody that binds to the primary antibodies, then wash.

Add a chemiluminescent substrate that reacts with the enzyme and emits light.

Detect the chemiluminescence signals corresponding to the NS3 and beta-actin bands, representing the presence and relative expression of the viral protein.

Following qPCR, resolve the cell lysate from the viral RNA transfected at 96 hours post transfection using SDS-PAGE. Then transfer the resolved proteins in the gel to a