Immunoblot Analysis of Apoptosis Inhibition in Host Cells Expressing an Antiapoptotic Bacterial Protein

Abstract

Source: Berens, C. et al. Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling. J. Vis. Exp. (2015)

This video demonstrates the use of immunoblotting to assess apoptosis inhibition by the bacterial effector protein CaeB in human cells. It outlines the steps for detecting apoptotic markers following doxycycline-induced expression of pro-apoptotic proteins in host cells expressing the effector.

Protocollo

1. Analysis of Host Cell Apoptosis by Immunoblot Analysis

1. Inspect cells prior to harvesting under the light microscope to check for cell death induction. Apoptotic cells are detectable by the presence of apoptotic bodies surrounding the dying cell.
2. Remove the supernatant and wash the adhered cells once with warm phosphate-buffered saline (PBS). Resuspend the cells in 100 µl of 2x Laemmli sample buffer, heat for 5 min at 95 °C, and store at -20 °C.
3. Load approximately 4 x 10⁴ cells per sample on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. Additionally, load 6 µl of a commercial protein ladder as a molecular weight marker. Run gels in a gel electrophoresis system under a constant voltage of 180 V for 45-60 min with 1x Laemmli running buffer.
4. Blot proteins onto polyvinylidene difluoride (PVDF) membranes (pore size of 0.45 µm) at a constant voltage of 16 V for 60 min using a Trans-Blot SD Semi-Dry Transfer Cell.
5. Block membranes in 10 ml of 5% nonfat dry milk in PBS/0.05% Tween 20 (MPT) for 1 hr at room temperature (RT).
6. Dilute 4 µl of anti-cleaved poly ADP-ribose polymerase (PARP) antibody in 4 ml of MPT and incubate O/N at 4 °C.
7. Perform three 10 min washing steps with 10 ml of PBS/0.05% Tween 20.
8. Incubate membranes with 0.8 µl horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml MPT.
9. After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 and detect horseradish peroxidase activity with a commercial kit according to the manufacturer’s protocol. Apply standard equipment for film development to visualize protein bands.
10. Remove bound antibodies by 30 min incubation at RT with 7 ml Western Blot Stripping Buffer.
11. After three washes with PBS/0.05% Tween 20, probe the membranes with 4 µl of anti-actin antibody in 4 ml of MPT O/N at 4 °C.
12. After three washing steps with MPT, incubate the membranes with 0.8 µl of horseradish peroxidase-conjugated secondary antibodies diluted in 4 ml of MPT.
13. After 1 hr incubation at RT, wash membranes three times with PBS/0.05% Tween 20 and detect horseradish peroxidase activity with a commercial kit according to the manufacturer’s protocol. Apply standard equipment for film development to visualize protein bands.
    NOTE: All incubation steps were performed on a plate shaker for the indicated time and temperature.

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
Doxycycline	Sigma Aldrich	D9891
Trans-Blot SD Semi-Dry Transfer Cell	Bio-Rad	170-3940
PageRuler Prestained Protein Ladder	Thermo Scientific	26616
PVDF membrane	Millipore	IPVH00010
Anti-GFP	life technologies	A6455
Anti-cleaved PARP	BD Bioscience	611038
Mouse IgG (H+L)-HRPO	Dianova	111-035-062
ECL Western Blotting Substrate	Thermo Scientific	32106