In vivo BioID protein purification
Shiyi Wang
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Abstract
In vivo BioID protein purification
Steps
Animal Preparation - Breed genotype-matched animals (wild-type C57BL6 or LRRK2 G2019Ski/ki) to produce single-genotype litters. - For each genotype (WT or G2019S), prepare 6 pups for injection.
AAV Injection - Inject 1 µL of AAVs carrying Astro-Ezrin-BioID (PHP.eB.GfaABC1D-Ezrin WT-BioID2-HA) or Astro-CYTO-BioID (PHP.eB.GfaABC1D-BioID2-HA) bilaterally into the cortex of P0-P2 mouse pups using a Hamilton syringe. - Monitor pups until they recover on a heating pad.
Biotin Injection - At P18, P19, and P20, subcutaneously inject biotin at 24 mg/kg to increase biotinylation efficiency.
Tissue Collection - At P21, remove the cerebral cortices and store at -80°C. - Pool 2 genotype-matched cortices at the time of protein isolation, yielding 3 independent replicates per BioID construct.
Protein Purification - Lyse each cortex in a buffer containing: - 50 mM Tris/HCl, pH 7.5 - 150 mM NaCl - 1 mM EDTA - Protease inhibitor mixture (Roche) - Phosphatase inhibitor mixture (PhosSTOP, Roche) - Add an equal volume of buffer containing: - 50 mM Tris/HCl, pH 7.5 - 150 mM NaCl - 1 mM EDTA - 0.4% SDS - 2% TritonX-100 - 2% deoxycholate - Protease inhibitor mixture - Phosphatase inhibitor mixture
Sonication and Centrifugation - Sonicate samples. - Centrifuge at 15,000 g for 10 minutes. - Ultracentrifuge the supernatant at 100,000 g for 30 minutes at 4°C.
Sample Preparation for Protein Binding - Add SDS detergent to the samples and heat at 45°C for 45 minutes. - Cool on ice.
Protein Binding - Incubate each sample with High-Capacity Streptavidin Agarose beads (ThermoFisher) at 4°C overnight.
Bead Washing - Wash beads serially:
Twice with a solution containing 2% SDS.
Twice with a buffer containing 1% TritonX-100, 1% deoxycholate, and 25 mM LiCl.
Twice with 1 M NaCl.
Five times with 50 mM ammonium bicarbonate.
Protein Elution - Elute biotinylated proteins attached to the agarose beads in a buffer containing: - 125 mM Tris/HCl, pH 6.8 - 4% SDS - 0.2% β-mercaptoethanol - 20% glycerol - 3 mM biotin - Heat at 60°C for 15 minutes.
Downstream Analysis - Subject the 12 total samples (3 per genotype per construct) to LC-MS/MS and downstream analysis.
