Feb 07, 2025
Validation of phosphor-ERM Antibody Specificity in WT and LRRK2 G2019S Knock-In(ki/ki) Mouse Brain Sections
- 1Duke University
Protocol Citation: Shiyi Wang 2025. Validation of phosphor-ERM Antibody Specificity in WT and LRRK2 G2019S Knock-In(ki/ki) Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrn6xg5b/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: February 05, 2025
Last Modified: February 07, 2025
Protocol Integer ID: 119635
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Chan Zuckerberg Initiative, Neurodegeneration Challenge Network
Grant ID: DAF2018-191999
Chan Zuckerberg Initiative, Neurodegeneration Challenge Network
Grant ID: DAF2021-237435
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Abstract
To confirm the specificity of the antibody used to detect phosphorylated ERM (pERM) in immunostaining by using Lambda protein phosphatase treatment on brain sections from both wild-type (WT) and LRRK2 G2019Ski/mice. Lambda protein phosphatase dephosphorylates proteins, which should eliminate phospho-ERM staining if the antibody is specific to the phosphorylated form of ERM.
Objective
Objective
To confirm the specificity of the antibody used to detect phosphorylated ERM (pERM) in immunostaining by using Lambda protein phosphatase treatment on brain sections from both wild-type (WT) and LRRK2 G2019Ski/ki mice. Lambda phosphatase dephosphorylates proteins, which should eliminate phospho-ERM staining if the antibody is specific to the phosphorylated ERM.
Materials
Materials
WT and LRRK2 G2019Ski/ki mouse brain sections
Reagents
Lambda Protein phosphatase enzyme and buffer: https://www.neb.com/en-us/products/p0753-lambda-protein-phosphatase-lambda-pp?srsltid=AfmBOoqA4jK4leWqMweJTU25CO0o4dyd5r3o11Mx9aN49mr1KlCzIcXC
Preparation of Brain Sections
Preparation of Brain Sections
Harvest brain tissues from WT and LRRK2 G2019Ski/ki mice (age matched)
Fix brain tissues in 4% paraformaldehyde (PFA) for 24 hours at 4 degrees.
Cryoprotect tissues in 30% sucrose solution until fully equilibrated
Embed and section brains using a cryostat to obtain 30um thick coronal sections, and store sections at -20 degrees
Treatment Conditions
Treatment Conditions
Divide brain sections from both WT and LRRK2 G2019Ski/ki mice into two groups
Lambda Protein Phosphatase Treatment Group: Treat sections with Lambda Protein phosphatase to dephosphorylate phospho-ERM
Control Group: Incubate sections under identical conditions but without Lambda Protein Phosphatase
Lambda Protein Phosphatase Treatment
Lambda Protein Phosphatase Treatment
Prepare Lambda Protein Phosphatase solution according to manufacturer instructions.
Incubate sections from the treatment group in the Lambda Protein phosphatase solution for 2 hours at 30°C.
Wash sections thoroughly with TBS to remove residual enzymes.
Immunostaining
Immunostaining
Block all sections in blocking solution (e.g., 5% normal goat serum and 0.3% Triton X-100 in PBS) for 1 hour at room temperature.
Incubate sections overnight at 4°C with the primary antibody targeting pERM at 1:100.
Wash sections with TBS three times.
Incubate sections overnight at 4°C with the secondary antibody
Wash with TBS and mount with STED mounting medium.
Imaging and Data Acquisition
Imaging and Data Acquisition
Image all sections using Olympus FV3000, Capture regions of interest within each section