Jun 26, 2025
Version 2
FLAG-tag mediated co-immunoprecipitation of phosphorylated Rab GTPases with cellular interacting proteins V.2
- Eve Napier1,
- Pawel Lis2,
- Dario Alessi2,
- Amir Khan1
- 1School of Biochemistry and Immunology, Trinity College Dublin;
- 2MRC PPU, School of Life Sciences, University of Dundee
- AKhanLab
Protocol Citation: Eve Napier, Pawel Lis, Dario Alessi, Amir Khan 2025. FLAG-tag mediated co-immunoprecipitation of phosphorylated Rab GTPases with cellular interacting proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvowjn9l4o/v2Version created by Amir Khan
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: June 25, 2025
Last Modified: June 26, 2025
Protocol Integer ID: 221081
Keywords: phosphorylated rab gtpase, tagged rab protein, rab protein, interacting proteins leucine rich repeat kinase, subset of rab gtpase, proteins leucine rich repeat kinase, rab gtpase, interactions between rab, phosphorylated flag, interacting protein, rab, protein
Funders Acknowledgements:
Research Ireland
Grant ID: 20/FFP-A/8446
The Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-000463
UK Medical Research Council
Grant ID: MC_UU_00018/1
Abstract
Leucine Rich Repeat Kinase 2 phosphorylates a subset of Rab GTPases at a conserved Ser/Thr residue in their switch 2 region. This modification disrupts interactions between Rabs and effectors/regulators while enabling binding to novel phospho-specific effectors. In this protocol, we use purified phosphorylated and non-phosphorylated FLAG tagged Rab proteins as 'bait' to immunoprecipitate interacting proteins. This is followed by mass spectrometry to identify proteins that bind preferentially to either phospho- or non-phospho Rabs.
Guidelines
Note on lysate preparation: Interacting proteins are immunoprecipitated from both A549 cell lysate and whole mouse brain lysate treated with the LRRK2 inhibitor MLi2. Lysates are prepared with cold lysis buffer freshly supplemented with cOmplete Mini (EDTA-free) protease inhibitor (Roche), PhosSTOP phosphatase inhibitor (Roche) and clarified by centrifuging at 17,000 x g for 15 min at 4°C. Protein concentrations are determined by Bradford Assay and the lysates stored at -80°C.
Materials
Buffers
- PBS-T: PBS supplemented with 0.01% Tween-20, 5mM MgCl2
- Lysis buffer: 50mM Tris-HCl pH 7.4, 150mM NaCl, 10% (w/v) glycerol, 10mM sodium-glycerophosphate, 10mM sodium pyrophosphate, 0.5% (v/v) NP40 - supplemented with cOmplete Mini (EDTA-free) protease inhibitor (Roche), PhosSTOP phosphatase inhibitor (Roche) and 5mM MgCl2
- High salt lysis buffer: lysis buffer supplemented with 500mM NaCl
Reagents
- Phosphorylated FLAG-Rab
- FLAG-Rab
- PBS (GIBCO brand)
- Pierce™ Anti-DYKDDDDK Magnetic Agarose (supplied as 25% slurry in PBS, 0.01% Tween-20 detergent, 0.02% sodium azide, pH 7.2 with a confirmed binding capacity of ~3.2mg FLAG Rab (~22kDa)/ml settled beads
- cOmplete Mini (EDTA-free) protease inhibitor (Roche)
- PhosSTOP phosphatase inhibitor (Roche)
Equipment
- Disc rotator
- Magentic seapration stand
Immunoprecipitation of Interacting Proteins
6 replicates are performed for each phospho-FLAG Rab and FLAG Rab, following the steps below for each replicate.
Aliquot 5μl settled volume of Pierce™ Anti-DYKDDDDK Magnetic Agarose in low binding Eppendorf tube and wash with 500μl PBS-T on a rotator for 5 minutes.
Place tube in magnetic separation stand and remove supernatant. Repeat washing step 3 times.
Add 20μg of FLAG protein to the washed beads and make up the volume to 500μl with PBS-T. Incubate at 4°C on a rotator for 1 hour.
Place tube in magnetic separation stand and remove supernatant without disturbing the bead pellet.
Wash the beads with 500μl PBS-T twice and remove supernatant completely.
Add cell lysate (~400μg A549 lysate or ~750μg brain tissue lysate) to the beads on ice and make up the volume to 500μl with cold lysis buffer. Incubate on a rotator for 1 hour at 4°C.
Note
Note on lysate preparation: Interacting proteins are immunoprecipitated from both A549 cell lysate and whole mouse brain lysate treated with the LRRK2 inhibitor MLi2. Lysates are prepared with cold lysis buffer freshly supplemented with cOmplete Mini (EDTA-free) protease inhibitor (Roche), PhosSTOP phosphatase inhibitor (Roche) and clarified by centrifuging at 17,000 x g for 15 min at 4°C. Protein concentrations are determined by Bradford Assay and the lysates stored at -80°C until required for the IP.
On a magnetic stand, remove supernatant and wash twice with 500μl cold high salt lysis buffer followed by twice with 500μl cold lysis buffer.
Note
Ensure supernatant is removed completely after the final wash.
Store samples at -80°C for mass spectrometry analysis or immunoblotting.