Jul 10, 2024
Protein extraction and Western blotting
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Protein extraction and Western blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj784lx1/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: July 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103174
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Protein extraction and Western blotting
**Prepare Membrane Solubilization Buffer** - 25 mM Tris (pH 7.4) - 150 mM NaCl - 1 mM CaCl2 - 1 mM MgCl2 - 0.5% NP-40 - Protease inhibitors
**Wash Cultured Astrocytes** - Wash cultured rat astrocytes twice with ice-cold TBS containing 1 mM CaCl2 and 1 mM MgCl2.
**Extract Protein** - Incubate astrocytes on ice in membrane solubilization buffer for 20 minutes with occasional agitation.
**Collect Cell Lysates** - Vortex briefly. - Centrifuge at 4°C at high speed for 10 minutes to pellet non-solubilized material.
**Store Supernatant** - Collect the supernatant and store at −80°C.
**Determine Protein Concentration** - Use the Pierce BSA Protein Assay Kit (Thermo Fisher).
**Prepare Protein Samples** - Mix lysates with 4x Pierce™ Reducing Sample Buffer (Thermo Scientific). - Incubate at 45°C for 45 minutes to denature proteins.
**Load Protein into Gels** - Load 7-10 μg of protein (cultured astrocyte lysates) into Bolt™ 4–12% Bis-Tris Plus gels (Thermo Scientific). - Run at 150 V for 1 hour.
**Transfer Proteins to Membrane** - Transfer proteins at 100 V to PVDF membrane (Millipore) for 1.5 hours.
**Block Membrane** - Block in 5% BSA in TBST (137 mM NaCl, 2.68 mM KCl, 24.7 mM Tris-Base, 0.1% (w/v) Tween 20) for 1 hour.
**Incubate with Primary Antibodies** - Incubate overnight at 4°C with the following primary antibodies: - Anti-LRRK2 (Rabbit, 1:500; ab133474, Abcam) - GAPDH (Mouse, 1:5000; ab8245, Abcam) - β-actin (Mouse, 1:5000; A5441, Millipore Sigma)
**Wash Membranes** - Wash membranes with TBST.
**Incubate with Secondary Antibodies** - Incubate in HRP secondary antibodies (Thermo Fisher Scientific) for 2 hours.
**Wash and Image Membranes** - Wash membranes in TBST. - Image on a Biorad Gel Doc imaging system.
**Quantify Protein Levels** - Quantify protein levels using FIJI software.