Nov 04, 2024
Western blot analysis
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen, Loukia Parisiadou 2024. Western blot analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7mr98gwz/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: November 04, 2024
Last Modified: November 04, 2024
Protocol Integer ID: 111531
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
In order to examine the link between LRRK2 activity and motor behaviors sensitive to D2R signaling, we evaluated whether the effects of blocking D2R signaling by haloperidol are influenced by LRRK2 inhibition. Haloperidol, a typical antipsychotic used to treat psychosis, is known to cause Parkinsonian-like side effects, including catalepsy in rodents and dyskinesia in humans. We hypothesized that if LRRK2 kinase activity plays a significant role in D2R signaling in the striatum, its inhibition may interfere with the cataleptic response induced by haloperidol.
Sample preparation
Sample preparation
After receiving the drug treatments striatal tissues were extracted and homogenized in 1x cell lysis buffer (Cell Signaling Technologies) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) using pellet pestles(30 seconds)
Protein concentration was determined by BCA protein assay (Thermo Scientific)
Western blot
Western blot
30 μg of total lysate were separated by 4–12% NuPage Bis-Tris PAGE (Invitrogen) and transferred to membranes using the iBlot nitrocellulose membrane Blotting system (Invitrogen) by following manufacturer protocol
The membranes were incubated with primary antibodies specific for phospho-Rab12 (1:1000, Abcam), total Rab12 (1:1000) Proteintech and β-actin(1:3000, Sigma) at 4C overnight.
The membranes following probing with secondary anti-mouse or and anti-rabbit antibodies (1:2000, Thermo Scientific) for 1 hour at room temperature
The membranes were incubated with Immobilon ECL Ultra Western HRP Substrate (Millipore) for 3 min prior to image acquisition
Chemiluminescent blots were imaged with iBright CL1000 imaging system (Thermo Fisher Scientific).
Western blot analysis
Western blot analysis
Images acquired with iBright CL1000 imaging system were uploaded to ibright analysis software.
The ibright analysis software detected the areas of interest automatically and manually adjusted them if needed.
Absolute volume and intensity were detected. Further statistical analyses were done using GraphPad
Prism 10.1 software