Protein extraction, quantification, and western blot for Bodo saltans

Dissolve pellet in lysis buffer2: 150 mM NaCl, 1% Triton X-100, 50 mM TrisHCl (pH8), 5 mM EDTA. Supplement lysis buffer with cOmplete Protease Inhibitor Coctail (Roche) (1 tablet per 10 ml of solution) and Benzonase nuclease (Sigma) immediately before extraction to prevent protein degradation and to decrease viscosity of extract, respectively.

Incubate extract on ice for 30 mins.

Centrifuge at 8000 × g to pellet debris.

Collect supernatant.

Quantify protein (eg. using the Pierce™ BCA Protein Assay Kit, (Thermo Fisher) according to the kit instructions).

Bodo saltans was cultured in a cerophyl-based medium enriched with 3.5 mM sodium phosphate dibasic (Na₂HPO₄)¹. Cultures were incubated at 22 °C in T25 tissue culture flasks containing 20 ml of media bacterized with Klebsiella pneumoniae subsp. Pneumoniae (ATCC® 700831).

Centrifuge 20 ml of 1 week old culture at 1200 × g for 12 mins at 19 °C to pellet the cells.

Dissolve pellet in lysis buffer²: 150 mM NaCl, 1% Triton X-100, 50 mM TrisHCl (pH8), 5 mM EDTA. Supplement lysis buffer with cOmplete Protease Inhibitor Coctail (Roche) (1 tablet per 10 ml of solution) and Benzonase nuclease (Sigma) immediately before extraction to prevent protein degradation and to decrease viscosity of extract, respectively.

Incubate extract on ice for 30 mins.

Centrifuge at 8000 × g to pellet debris.

Collect supernatant.

Quantify protein (eg. using the Pierce™ BCA Protein Assay Kit, (Thermo Fisher) according to the kit instructions).

For western blot, adjust protein concentrations so that they are equal between samples.

1. Separate protein extracts on polyacrylamide gels (eg. Bolt Bis-Tris Plus, Thermo Fisher).

2. Transfer proteins to PVDF membrane (eg. using mini blot module (Thermo Fisher)).

3. Block the membrane for 30 mins at room temperature in 5% skimmed milk diluted in wash buffer (PBS with 0.1% Tween20).

4. Incubate the membrane overnight at 4 °C with primary antibodies (1:100 times diluted in 5% milk in wash buffer).

5. Wash the membrane 3 × 10 mins with wash buffer (PBS with 0.1% Tween 20).

6. Incubate the membrane for 1 hour at room temperature with Horseradish Peroxidase (HRP)-labelled secondary antibodies (1:5000 dilution in 5% milk in wash buffer).

7. Wash the membrane 3 × 10 mins with wash buffer (PBS with 0.1% Tween 20).

8. Develop the signal with HRP substrate (eg. Immobilon western kit, Merk) and scan for chemiluminescent signal (eg. with ImageQuant LAS4000 or Bio Rad imaging system).

1. Gomaa Fatma, Li ZuHong, Docampo Roberto, Girguis Peter, E. V. Bodo saltans culture protocol V.2. Protocols.io (2018). doi:10.17504/protocols.io.sh6eb9e

2. Newton, I. L. G., Savytskyy, O. & Sheehan, K. B. Wolbachia Utilize Host Actin for Efficient Maternal Transmission in Drosophila melanogaster. PLOS Pathog. 11 , e1004798 (2015).