Sep 30, 2023

Public workspaceCryo-EM sample preparation for PI3KC3-C1~RAB1A complex

This protocol is a draft, published without a DOI.
  • 1University of California, Berkeley;
  • 2Aligning Science Across Parkinson's
Annan SI Cook
Open access
Protocol Citation: Annan SI Cook 2023. Cryo-EM sample preparation for PI3KC3-C1~RAB1A complex. protocols.io https://protocols.io/view/cryo-em-sample-preparation-for-pi3kc3-c1-rab1a-com-c2pcydiw
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
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
This protocol details cryo-EM sample preparation for PI3KC3-C1~RAB1A complex.
Attachments
852-2199.pdf
852-2199.pdf
28KB
Materials
Materials

  • RAB1A(Q70L) protein
  • EDTA
  • GTP
  • PD-10 column
  • HEPES pH 7.5
  • NaCl
  • MgCl2
  • TCEP
  • PI3KC3-C1 protein
GTP Loading of RAB1A
GTP Loading of RAB1A
1h 20m
Strip the protein of its bound divalent cations by incubating RAB1A(Q70L) with Concentration5 millimolar (mM) EDTA at TemperatureRoom temperature for Duration00:30:00 .
30m
Incubation
Add a large molar excess (Concentration50 millimolar (mM) ) of GTP to the protein.

Incubate the mixture for an additional Duration00:30:00 to allow GTP loading.

30m
Incubation
Quench the reaction by adding Concentration20 millimolar (mM) MgCl2 at TemperatureRoom temperature for Duration00:20:00 .
20m
Pipetting
Desalting of GTP-loaded RAB1A
Desalting of GTP-loaded RAB1A
Purify the GTP-loaded RAB1A through a PD-10 column equilibrated with a final buffer containing Concentration25 millimolar (mM) HEPES Ph7.5 , Concentration150 millimolar (mM) NaCl, Concentration2 millimolar (mM) MgCl2, and Concentration25 millimolar (mM) TCEP.
Preparation of PI3KC3-C1~RAB1A Complex
Preparation of PI3KC3-C1~RAB1A Complex
1h
Prepare a 1:20 molar ratio of PI3KC3-C1 to RAB1A (Concentration0.55 micromolar (µM) PI3KC3-C1: Concentration11 micromolar (µM) RAB1A).
Incubate the mixture for Duration01:00:00 at TemperatureRoom temperature to allow complex formation.
1h
Incubation