Sep 30, 2023

Cryo-EM sample preparation for PI3KC3-C1~RAB1A complex

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo3odbv4o/v1
  • 1University of California, Berkeley;
  • 2Aligning Science Across Parkinson's
Annan SI Cook
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo3odbv4o/v1
Protocol Citation: Annan Cook 2023. Cryo-EM sample preparation for PI3KC3-C1~RAB1A complex. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3odbv4o/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: September 22, 2023
Last Modified: May 31, 2024
Protocol  Integer ID: 88516
Keywords: ASAPCRN, em sample preparation for pi3kc3, rab1a complex, pi3kc3, protocol details cryo, em sample preparation, cryo, preparation, c1, sample
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol details cryo-EM sample preparation for PI3KC3-C1~RAB1A complex.
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Materials
Materials

  • RAB1A(Q70L) protein
  • EDTA
  • GTP
  • PD-10 column
  • HEPES pH 7.5
  • NaCl
  • MgCl2
  • TCEP
  • PI3KC3-C1 protein
GTP Loading of RAB1A
1h 20m
Strip the protein of its bound divalent cations by incubating RAB1A(Q70L) with 5 millimolar (mM) EDTA at Room temperature for 00:30:00 .
30m
Add a large molar excess (50 millimolar (mM) ) of GTP to the protein.

Incubate the mixture for an additional 00:30:00 to allow GTP loading.

30m
Quench the reaction by adding 20 millimolar (mM) MgCl2 at Room temperature for 00:20:00 .
20m
Desalting of GTP-loaded RAB1A
Purify the GTP-loaded RAB1A through a PD-10 column equilibrated with a final buffer containing 25 millimolar (mM) HEPES 7.5 , 150 millimolar (mM) NaCl, 2 millimolar (mM) MgCl2, and 25 millimolar (mM) TCEP.
Preparation of PI3KC3-C1~RAB1A Complex
1h
Prepare a 1:20 molar ratio of PI3KC3-C1 to RAB1A (0.55 micromolar (µM) PI3KC3-C1: 11 micromolar (µM) RAB1A).
Incubate the mixture for 01:00:00 at Room temperature to allow complex formation.
1h