Jul 10, 2024
Protein extraction and Western blotting
- 1Duke University
Document Citation: Shiyi Wang 2024. Protein extraction and Western blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb44xvx1/v1
License: This is an open access document 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
Created: May 23, 2023
Last Modified: July 10, 2024
Document Integer ID: 82326
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
1. Protein was extracted from cultured rat astrocytes using membrane solubilization buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.5% NP-40, and protease inhibitors).
2. Cultured astrocytes were washed twice with ice-cold TBS containing 1 mM CaCl2and 1 mM MgCl2and incubated on ice in membrane solubilization buffer for 20 minutes with occasional agitation.
3. Cell lysates were collected, vortexed briefly, and centrifuged at 4°C at high speed for 10 minutes to pellet non-solubilized material. The supernatant was collected and stored at −80°C.
4. Pierce BSA Protein Assay Kit (Thermo Fisher) was used to determine protein concentration, and lysates were mixed with 4× Pierce™ Reducing Sample Buffer (Thermo Scientific) and incubated at 45°C for 45 minutes to denature proteins.
5. 7-10 μg (cultured astrocyte lysates) of protein was loaded into Bolt™ 4–12% Bus-Tris Plus gels (Thermo Scientific) and run at 150 V for 1 hour.
6. Proteins were transferred at 100 V to PVDF membrane (Millipore) for 1.5 hours, blocked 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 and incubated in primary antibodies overnight at 4°C.
7. Primary antibodies used were: anti-LRRK2 (Rabbit, 1:500; ab133474, abcam), GAPDH (mouse, 1:5000; ab8245, abcam), β-actin (mouse, 1:5000; A5441, Millipore Sigma), phospho-ERM (Rabbit, 1:500; #3141, Cell Signaling), ERM (Rabbit, 1:500; #3142, Cell Signaling), phospho-Rab10 (Rabbit, 1:500; ab230261, abcam), Rab10 (Rabbit, 1:500; ab237703, abcam).
8. The next day, membranes were washed with TBST, incubated in HRP secondary antibodies (Thermo Fisher Scientific) for 2 hours, washed in TBST, and imaged on a Biorad Gel Doc imaging system.
9. Protein levels were quantified using FIJI.