Apr 24, 2026
Version 2
Western blot and phostag gels V.2
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen, Loukia Parisiadou 2026. Western blot and phostag gels. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkrxd5v5r/v2Version created by Chuyu Chen
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: April 24, 2026
Last Modified: April 24, 2026
Protocol Integer ID: 315711
Keywords: ASAPCRN, dopaminergic neuronal release site, neuronal release site, evoked dopamine release, dopamine release in the nigrostriatal pathway, dopaminergic neuron, dopamine release, rab3a with various active zone protein, organization of presynaptic release site, lrkk2 kinase activity, changes in lrkk2 kinase activity, phosphorylated rab3a, various active zone protein, presynaptic release site, kinase activity, dopaminergic, dopamine, mouse brain, organization of active zone, cellular basis for the observed deficit, axonal varicosity, several pathway, structural disorganization of active zone, active zone
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
Dopaminergic neuronal release sites have unique characteristics, as only about 25–30% of axonal varicosities contain active zones that support transmitter release. Rim1/2 proteins are important scaffolding molecules that organize these release sites, and a dopaminergic neuron-specific knockout of Rim1/2 leads to the structural disorganization of active zones and almost completely abolishes evoked dopamine release in the nigrostriatal pathway.
Our phosphoproteomic analysis showed that several pathways associated with the organization of presynaptic release sites are altered with changes in LRKK2 kinase activity, and we found a decrease in the interaction of phosphorylated Rab3a with various active zone proteins from mouse brain. Therefore, we next examined whether alterations in the number and/or organization of active zones may form the cellular basis for the observed deficits in dopamine release in Lrrk2G2019S mice.
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Brain extracts
Homogenize tissue in 1x cell lysis buffer (Cell Signaling Technologies) supplemented with the 1x Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) using
pellet pestles for 30 seconds
Centrifuge at 13800 g for 10 min at 4℃. Keep the supernatant.
Determine protein concentration using BCA Protein Assays (Thermo Scientific).
Separated equal amounts of protein (30ug) by 4–12% NuPAGE Bis-Tris PAGE (Invitrogen)
Transfer to membranes using the iBlot nitrocellulose membrane blotting system (Invitrogen) according to the manufacturer's protocol
Synaptosomes
Homogenize mouse striata in 1ml iced-cold homogenizing buffer using a Teflon homogenizer (15 strokes).
homogenizing buffer recipe ( 4mM HEPES, 320mM Sucrose and Halt protease supplemented with phosphatase inhibitor cocktail (Thermo Fisher Scientific))
Centrifuge at 1000 g for 10 min at 4℃.
Centrifuge supernatant (S1) at 12,500 g for 15 min at 4°C.
Remove supernatant (S2)
Re-homogenize the pellet (P2) in 1 ml homogenizing buffer with 10 strokes in a Teflon homogenizer.
Add P2 homogenate to the top of a sucrose gradient made of 5ml 1.2M sucrose and 5ml 0.8M sucrose, and centrifuge at 69,150 g (SW41) for 70 min at 4°C.
The synaptic plasma membrane fraction (SPM) in the interphase between two sucrose fractions is collected using a syringe and transfer to clean ultracentrifuge tubes
centrifuge in a swinging bucket rotor (SW55) at 200,000 g for 30 min at 4°C.
Resuspend the pellet with SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris) supplemented with the Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific)
Use BCA Protein Assays (Thermo Scientific) to determine protein concentration
separate equal amounts of protein (5ug) by 4–12% NuPAGE Bis-Tris PAGE (Invitrogen)
Transfer to membranes using the iBlot nitrocellulose membrane blotting system (Invitrogen) according to the manufacturer's protocol
Phos Tag gels
Homogenize SNc tissues in EDTA-free Lysis bufferusing pellet pestles (30 seconds), supplement with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific).
EDTA-free Lysis buffer recipe (1% Triton X-100, 1% glycerol, 150 mM NaCl, 25 mM HEPES, 1.5 mM MgCl2)
Centrifuge at 13800 g for 10 min at 4℃. Keep the supernatant.
Use BCA Protein Assays (Thermo Scientific) to determine protein concentration
Separate 10 μg of lysa teby 12.5% SuperSepTM Phos-tagTM (Wako).
Wash gels with 1x TG transfer buffer (Fisher BioReagents) supplemented with 10 % Methanol and 10 mM EDTA, 10 minutes x 3
Incubate gels with 1x TG transfer buffer with 10% Methanol for 10 minutes
Transfer gels to Nitrocellulose membranes (Thermo Scientific) using the Mini Gel Tank and Blot Module (ThermoFisher) at 15V for 3 hours
Image acquisition
Incubate the membranes in 3% BSA in 1x Tris buffered saline (Sigma) supplemented with 0.1% Tween 20 for 1 hour
Incubate the membranes with primary antibodies specific for LRRK2/Dardarin clone
N137/6 (1:1000, NeuroMab), LRRK2/Dardarin clone N241A/34 (1:1000, NeuroMab), Th (1:1000, Millipore), Rab3a (1:1000, Sigma), Rab3c (1:1000, Proteintech), synaptophysin (1:1000, Cell Signaling), Vmat2 (1:1000, R&D Systems), Snap25 (1:1000, Proteintech), DAT (1:1000, Millipore), pS1292 LRRK2 (1:100, Abcam), pS935 LRRK2 (1:100, Abcam), Rim1 (1:1000, Proteintech), GDI1 (1:1000, Sigma), GDI2 (1:1000, Novus), phospho-Rab12 (1:1000, Abcam), total Rab12 (1:1000, Proteintech), and β-actin (1:3000, Sigma) overnight at 4°C.
Wash membranes with TBS/Tween-20, 10 mins x3
Incubate the membranes with secondary anti-mouse or anti-rabbit antibodies (1:2000, Thermo Scientific) for 1 hour at room temperature.
Wash membranes with TBS/Tween-20, 10 mins x3
Incubate the membranes with Immobilon ECL Ultra Western HRP Substrate (Millipore) for 3 minutes RT
Chemiluminescent blots were imaged using the iBright imaging system (Thermo Fisher Scientific).