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

Abstract

Source: Gilmore, S. F. et al. Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development. J. Vis. Exp. (2022)

This video demonstrates the use of Western blotting to evaluate the structural integrity of the major outer membrane protein (MOMP) within nanoparticle-based vaccine complexes. It outlines the key steps involved in protein transfer, antibody labeling, and fluorescence imaging.

Protocol

1. Western blot

1. For western blotting, resolve the major outer membrane protein (MOMP)– telodendrimer nanolipoprotein particles (tNLPs) by SDS-PAGE and transfer the gels using a commercial dry blotting system with standard settings according to the manufacturer’s protocol.
   1. Remove the blots from the stack after the transfer is complete, and incubate each blot overnight at 4 °C in a suitable blocking buffer containing 0.2% Tween 20 and either 0.5 mg/mL monoclonal antibody (MAb) 40 or 0.2 mg/mL MAbHIS anti-His-tag antibody directed against the His-tag from Δ49ApoA1 protein.
      NOTE: The antibody dilutions used for blotting are 1:1,000 for MAb40 and 1:500–1,000 for MAbHIS antibody.
   2. Wash each blot 3 times for 5 min with PBS-T (1x phosphate buffered saline (PBS), 0.2% Tween 20, pH 7.4).
   3. Incubate the blots for 1 h in blocking buffer containing secondary antibody conjugated to a fluorophore (e.g., Infrared dye (IRDye)) at a 1:10,000 dilution.
   4. Rewash the blots 3 times for 5 min with PBS-T. Use a fluorescence imager to image the blots after the final wash.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
iBlot+2:22 Dry Blotting System	Life Technologies
iBlot 2 Transfer Stacks, PVDF	Life Technologies	IB24001
Image Studio V2.0 software	Li-COR Biiosciences
Immun-Blot PVDF Membrane	Bio-Rad	1620177
LI-COR Odyssey Fc imager	Li-COR Biiosciences
Odyssey Blocking Buffer in TBS containing 0.2% Tween 20	Li-COR Biosciences	927-50000
Orbital Shaker
PBS-T (1x PBS, 0.2% Tween 20, pH 7.4)
Primary antibody: MAb40 (monoclonal antibody to the variable domain 1 (VD1) of C. muridarum MOMP
Primary antibody: MAbHIS, Penta-His antibody	Qiagen	34660
Secondary antibody: IRDye 800CW goat (polyclonal) anti-mouse IgG (heavy and light)	Li-COR Biosciences	926-32210