Jul 10, 2024
In vivo BioID protein purification
- 1Duke University
Protocol Citation: Shiyi Wang 2024. In vivo BioID protein purification. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r3z6g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103177
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
In vivo BioID protein purification
**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.