Dec 30, 2025
SDS and Clear Native Gel Electrophoresis
- Akio Mori1,
- Robert Edwards1
- 1University of California San Francisco
Protocol Citation: Akio Mori, Robert Edwards 2025. SDS and Clear Native Gel Electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyqydg5r/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 20, 2025
Last Modified: December 30, 2025
Protocol Integer ID: 233097
Keywords: synuclein, proteins from mouse brain sample, clear native gel electrophoresi, clear native gel electrophoresis this protocol, mouse brain sample, sd, related protein
Abstract
This protocol describes SDS–PAGE and clear native PAGE procedures for analyzing α-synuclein, Gba, and related proteins from mouse brain samples.
Materials
Materials and Reagents
- Gels 4–20% precast SDS–polyacrylamide gels (Bio-Rad, 5671095)
4–16% Bis-Tris Native Gels (Thermo, BN1002)
- Sample Buffers NativePage Sample Buffer (Thermo, BN2003)
- SDS-PAGE Running Buffer
25 mM Tris, 192 mM Glycine, 0.1% (w/v) SDS
- Transfer Materials Nitrocellulose membranes, 0.22 μm (1620115, Bio-Rad)
Transfer buffer 1 L
| A | B | |
| Transferbuffer 10× | 100 mL | |
| Methanol | 200 mL | |
| DW | 700 mL |
- CBB staining solution
Coomassie brilliant blue (Bio-Rad, 161-0406)
- Standards SDS-PAGE: Precision Plus Protein Standards (Bio-Rad, 161-0363)
Native-PAGE: NativeMark protein standard (Invitrogen, LC0725)
- Blocking and Wash Buffers 5% nonfat dry milk in TBS-T (0.05% Tween-20)
- Secondary antibodies IRDye 680LT or 800CW (LI-COR Biosciences)
HRP-conjugated secondaries (for ECL detection)
- Other Reagents 4% paraformaldehyde (PFA)
- ECL-based Detection
ECL Prime Western Blotting Detection Reagents (Cytiva, RPN2232)
High Performance chemiluminescence film (Cytiva, 28906836)
Troubleshooting
SDS–PAGE
Prepare protein samples and load them onto 4–20% precast SDS–polyacrylamide gels (Bio-Rad, 5671095).
Run electrophoresis following standard SDS–PAGE procedures.
Start at 80 V for approximately 30 minutes until the stacking is complete, then increase the voltage to 90–110 V to continue the separation.
Transfer proteins to 0.22 μm nitrocellulose membranes at 100 V for 1 h at 4°C.
(Optional for α-synuclein monomer detection)
Pretreat membranes with 4% PFA for 30 min before blocking.
Block membranes in 5% nonfat dry milk/TBS-T (0.05% Tween-20).
Incubate membranes with primary antibodies in 5% nonfat dry milk/TBS-T overnight at 4°C with gentle agitation.
Wash 3× 10 min with TBS-T.
Incubate with IRDye secondary antibodies for 1 h at room temperature.
Wash 3× 10 min with TBS-T.
Visualize and quantify signals using the Odyssey infrared imaging system.
Clear Native PAGE
Prepare samples in NativePage Sample Buffer.
Load samples onto 4–16% Bis-Tris native gels.
Run native PAGE at a constant 150 V for 2 h.
- Cathode buffer: 50 mM Tricine, 15 mM Bis-Tris, pH 7.0
- Anode buffer: 50 mM Bis-Tris, pH 7.0
Include NativeMark as the molecular weight standard.
Transfer proteins to 0.22 μm nitrocellulose membranes at 100 V for 1 h at 4°C.
Visualize standards on the gel using CBB staining.
Proceed with immunoblotting:
- Block membranes in 5% milk/TBS-T
- Incubate with primary antibodies overnight at 4°C
- Wash and incubate with IRDye-conjugated secondary antibodies for 1 h at room temperature
- Detect signal using the Odyssey imaging system
ECL-based Detection of Endogenous Gba
Incubate the membrane with primary antibody (diluted in 5% milk/TBS-T) overnight at 4°C with gentle agitation.
Wash 3× 10 min with TBS-T.
Incubate with HRP-conjugated secondary antibodies (diluted in 5% milk/TBS-T) for 1 h at room temperature with gentle agitation.
Wash 3× 10 min with TBS-T.
Incubate the membrane with Western Blotting Detection Reagents for 5 min before detection.
Expose onto chemiluminescence film or use a digital imaging system for signal detection.