TITLE: Identification of interacting proteins using proximity-dependent biotinylation with BioID2 in Trypanosoma brucei
AUTHORS: Jan Pyrih, Julius Lukeš

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A range of recently developed techniques that take advantage of proximity biotinylation, such as BioID, TurboID, and APEX, are particularly suitable for the studies of protein-protein interactions. As compared to classical co-immunoprecipitation, their advantage is higher reproducibility and capacity to identify stable complexes and also capture transient interactions. Briefly, the protein of interest is fused with modified biotin ligase, which promiscuously biotinylates proteins in its proximity. However, until now BioID has been used in</span><span style = "font-style:italic;"> T. brucei</span><span> only in just a handful of studies. Here, we took advantage of the recently developed advanced biotin ligation-based approach named BioID2, which we have successfully adapted for </span><span style = "font-style:italic;">T</span><span>. </span><span style = "font-style:italic;">brucei</span><span>. In contrast to the classical BioID technique, BioID2 biotin ligase is smaller (26 kDa), more specific, and attached to the protein of interest by several nm-long linker arm, which improves protein folding and the biotinylation range. When applied to </span><span style = "font-style:italic;">T. brucei</span><span>, BioID2 labeling produced a highly specific output. </span></div></div>

[STEPS]
?. [Hypotonic mitochondria isolation]
resuspend cell pellets in 50 ml of SBG and spin again at same condition
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resuspend cell pellet in 3 ml of DTE
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Add 0,5 ml of 60% Sucrose, mix the sample in dounce and transfer it into 3 eppendorf tubes
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Spin down: 16,000x g, 10min, 4°C
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Resuspend all 3 pellets in 1,5 ml of STM (merge everything in 2 ml eppendorf tubes)
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Pipette MgCl2 to a final concentration 5 mM, CaCl2 to a  final concentration 0.5 mMand DNase  to a  final concentration 5ug/ml
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Incubate on ice for 20 minute
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STE Wash 1,5 ml, 3x, 12,000x g, 10 min, 4°C
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Resuspend pellets in 1,8 ml of Boiling Buffer, 10 minutes, 80°C
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Spin down: 16,000x g, 10min, 20°C
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Meanwhile wash 50 ul of dynabeads per sample 2x times with 1ml of Incubation buffer, each time 5 minutes
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Mix supernatants from step 14 with 18 ml of Incubation Buffer (supplemented with ROCHE Complete protease inhibitors EDTA free)
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Add 50 uL of washed dynebeads resuspended in incubation buffer from step 15 per sample (final 0.5 mg per sample) (Dynabeads™ MyOne™ Streptavidin C1 -10 mg/ml)
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Roll for 2 hours at RT at falcon tubes, then place overnight at fridge
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Wash the beads 3 times with 1,5 ml of Boiling buffer
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Wash the beads 3 times with 1,5 ml of Incubation buffer
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Remove all liquid, put dry dynabeads into to the -80 freezer
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Spin down  3 x109 cells for each replicate – 1500x g, 15min, 4°C
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Dounce – 10 strokes
?. [Prior steps - cloning]
Tag you protein of interest with linker_BioID2 tag sequence according to attached pptx file
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SBG recipe:150 mM NaCl20 mM Glucose20 mM NaH2PO4pH 7.9
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DTE recipe:TRIS 1 mMEDTA 1 mMpH 8.0
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STM recipeSucrose 250 mMTris, pH 8.0  20 mMMgCl2    2mM
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snap freeze with liquid nitrogen and store in -80°C until needed or proceed directly to Dynabeads protein isolation
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Boiling buffer recipe:TRIS 50 mMEDTA 1mMSDS 1%pH 7.4
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Incubation buffer recipe:NaCl  150 mMTRIS 50 mMEDTA 5mMTRITON X-100 1%pH 7.4
?. [Biotin treatment]
Supplement SDM-79 media with 100 uM Biotin over night
?. [Prior steps - cloning]
Screen for a successful biotinylation in the cells supplemented with biotin on IFA using fluorescent Alexa-488-streptavidin