Western Blot for Assessing the Structural Integrity of a Bacterial Vaccine Antigen

Take an SDS-PAGE gel containing protein bands from membrane protein-nanolipoprotein particles. These complexes carry the Chlamydia oligomeric membrane protein or MP, a promising vaccine antigen against Chlamydia infection.

Use protein bands from purified oligomeric MP as the control.

Assemble the gel, western blot membrane, and blotting components in a dry blotting system.

Apply an electric current to transfer the resolved proteins onto the membrane.

Remove the membrane and incubate it in a buffer containing detergents and primary antibodies.

The detergents block non-specific interaction sites, while the primary antibodies target the MP.

Wash the membrane to remove unbound primary antibodies.

Incubate with fluorophore-conjugated secondary antibodies targeting the primary antibodies.

Wash the membrane to remove excess secondary antibodies.

Capture fluorescence images of the membrane.

Molecular bands matching the control oligomeric MP confirm the structural integrity of the MP within the complexes, supporting its antigenic potential.

Resolve the samples by SDS-PAGE and transfer the gel stacks using a commercial dry blotting system for Western blot analysis. Remove the blots from the stack and