TITLE: Protein extraction, quantification, and western blot for Bodo saltans
AUTHORS: Ewa  Chrostek,   Mastaneh Ahrar,   Gregory Dd Hurst

[DESCRIPTION]
This protocol is used in our Laboratory in Liverpool to work with proteins from Bodo saltans and other kinetoplastids (eg. trypanosomes). 

[STEPS]
SECTION: Culture conditions
1. 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). 
SECTION: Protein extraction and quantification
26. 
SECTION: Protein extraction and quantification
1.1. Centrifuge 20 ml of 1 week old culture at 1200 × g for 12 mins at 19 °C to pellet the cells.
SECTION: Protein extraction and quantification
0.26. 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.
SECTION: Protein extraction and quantification
0.26. Incubate extract on ice for 30 mins.
SECTION: Protein extraction and quantification
0.26. Centrifuge at 8000 × g to pellet debris.
SECTION: Protein extraction and quantification
0.26. Collect supernatant.
SECTION: Protein extraction and quantification
0.26. Quantify protein (eg. using the Pierce™ BCA Protein Assay Kit, (Thermo Fisher) according to the kit instructions).
SECTION: Protein extraction and quantification
1.2. 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. 
SECTION: Protein extraction and quantification
1.3. Incubate extract on ice for 30 mins.
SECTION: Protein extraction and quantification
1.4. Centrifuge at 8000 × g to pellet debris.
SECTION: Protein extraction and quantification
1.5. Collect supernatant.
SECTION: Protein extraction and quantification
1.6. Quantify protein (eg. using the Pierce™ BCA Protein Assay Kit, (Thermo Fisher) according to the kit instructions).
SECTION: Western blot
2. For western blot, adjust protein concentrations so that they are equal between samples. 
SECTION: Western blot
2.1. 1. Separate protein extracts on polyacrylamide gels (eg. Bolt Bis-Tris Plus, Thermo Fisher).
SECTION: Western blot
2.2. 2. Transfer proteins to PVDF membrane (eg. using mini blot module (Thermo Fisher)). 
SECTION: Western blot
2.3. 3. Block the membrane for 30 mins at room temperature in 5% skimmed milk diluted in wash buffer (PBS with 0.1% Tween20).
SECTION: Western blot
2.4. 4. Incubate the membrane overnight at 4 °C with primary antibodies (1:100 times diluted in 5% milk in wash buffer). 
SECTION: Western blot
2.5. 5. Wash the membrane 3 × 10 mins with wash buffer (PBS with 0.1% Tween 20). 
SECTION: Western blot
2.6. 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). 
SECTION: Western blot
2.7. 7. Wash the membrane 3 × 10 mins with wash buffer (PBS with 0.1% Tween 20). 
SECTION: Western blot
2.8. 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). 
SECTION: Western blot
2.9.   

SECTION: References
3. 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).