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

JoVE Encyclopedia of Experiments

Microbiologie

0 weergaven • 2:44 min • October 30th, 2025

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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 incubate them overnight at four degrees Celsius in a blocking buffer containing 0.2% TWEEN 20 and 0.5 micrograms per milliliter MAb40, or 0.2 micrograms per milliliter MAbHIS antiHIS-tag antibody directed against the HIS-tag from delta-49ApoA1 protein.

Wash each blot with PBST three times for five minutes per wash. Incubate the blots for one hour in a blocking buffer containing secondary antibody conjugated to a fluorophore in a dilution of 1 to 10,000. Repeat the washes with PBST and capture the image using a fluorescence imager after the final wash.

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Laatst bijgewerkt: 22 augustus 2026