Sep 01, 2025
SDS-PAGE and Western Blotting
- Jasmin Jansen1,2,3
- 1University of Konstanz, Department of Biology;
- 2Aligning Science Across Parkinson's;
- 3Konstanz Research School of Chemical Biology (KoRS-CB)
Protocol Citation: Jasmin Jansen 2025. SDS-PAGE and Western Blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6ywrdvqe/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: May 10, 2025
Last Modified: September 01, 2025
Protocol Integer ID: 218024
Keywords: ASAPCRN, Western blotting, SDS-PAGE, phosphorylation of lrrk1, rich repeat kinase, wide interactomes of leucine, rab gtpases by sd, phosphorylation activity, phosphorylation, rab gtpase, procedure to analyse protein abundance, analyse protein abundance, lrrk2, lrrk1, western blotting this protocol, proteome, leucine
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000519
Abstract
This protocol describes the sample preparation and procedure to analyse protein abundance and phosphorylation of LRRK1, LRRK2 and Rab GTPases by SDS-PAGE and Western Blotting.
It is meant to accompany the manuscript and methods section of "Probing the proteome-wide interactomes of Leucine-rich Repeat Kinases 1 and 2 and alterations in their phosphorylation activity".
Materials
Recombinant Anti-RAB7 (phospho S72) antibody [MJF-R38-1] (ab302494)AbcamCatalog #ab302494 Anti-RAB10 (phospho T73) antibody [MJF-R21]AbcamCatalog #ab230261 Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)]AbcamCatalog #ab133450 HRP-rabbit IgGJackson ImmunoResearch Laboratories, Inc.Catalog #111-035-003 PageRuler™ Prestained Protein Ladder 10 to 180 kDaThermo Fisher ScientificCatalog #26616
Spectra™ Multicolor High Range Protein LadderThermo FisherCatalog #26625
HRP-mouse IgGJackson ImmunoResearch Laboratories, Inc.Catalog #115-035-062
LRRK2 AntibodyCell Signaling TechnologyCatalog #5559
Recombinant Anti-RAB10 antibody [MJF-R23]AbcamCatalog #ab237703
Anti-RAB7 antibody [EPR7589] - Late Endosome MarkerAbcamCatalog #ab137029
Anti-RAB12 antibody [EPR28814-81]AbcamCatalog #ab316770
Recombinant Anti-RAB12 (phospho S106) antibody [MJF-R25-9]AbcamCatalog #ab256487
Monoclonal M2 antibody (anti-FLAG)Merck MilliporeSigma (Sigma-Aldrich)Catalog #F1804-200UG
Anti-alpha Tubulin antibody [DM1A] - Loading ControlAbcamCatalog #ab7291
anti-HA monoclonal antibodyLabcorp (formerly Covance)Catalog #MMS-101R
Protocol materials
cOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001
Recombinant Anti-RAB7 (phospho S72) antibody [MJF-R38-1] (ab302494)AbcamCatalog #ab302494
Anti-RAB12 antibody [EPR28814-81]AbcamCatalog #ab316770
Monoclonal M2 antibody (anti-FLAG)Merck MilliporeSigma (Sigma-Aldrich)Catalog #F1804-200UG
anti-HA monoclonal antibodyLabcorp (formerly Covance)Catalog #MMS-101R
Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)]AbcamCatalog #ab133450
HRP-mouse IgGJackson ImmunoResearch Laboratories, Inc.Catalog #115-035-062
Recombinant Anti-RAB12 (phospho S106) antibody [MJF-R25-9]AbcamCatalog #ab256487
Anti-alpha Tubulin antibody [DM1A] - Loading ControlAbcamCatalog #ab7291
Recombinant Anti-RAB10 antibody [MJF-R23]AbcamCatalog #ab237703
HRP-rabbit IgGJackson ImmunoResearch Laboratories, Inc.Catalog #111-035-003
LRRK2 AntibodyCell Signaling TechnologyCatalog #5559
Anti-RAB7 antibody [EPR7589] - Late Endosome MarkerAbcamCatalog #ab137029
Anti-RAB10 (phospho T73) antibody [MJF-R21]AbcamCatalog #ab230261
PageRuler™ Prestained Protein Ladder 10 to 180 kDaThermo Fisher ScientificCatalog #26616
Spectra™ Multicolor High Range Protein LadderThermo FisherCatalog #26625
Sample preparation
35m
Lyse frozen cell pellets (from 6-well dishes and 35 mm diameter) in 100 µL RIPA buffer (50 millimolar (mM) Tris-HCl pH 8, 150 millimolar (mM) NaCl, 0.5 Mass / % volume sodium deoxycholate, 0.1 Mass / % volume SDS, 1 % (v/v) NP-40, 1 millimolar (mM) DTT, 1x cOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001 ) with phosphatase inhibitors (5 millimolar (mM) NaF, 5 millimolar (mM) beta-glycerophosphate, 1 millimolar (mM) Na3VO4) on ice.
Incubate for 00:20:00 at 4 °C and gentle agitation.
20m
Pellet cell debris by centrifugation at 16000 x g for 00:10:00 and 4 °C .
10m
Take supernatant and determine protein concentration via BCA (bicinchoninic acid) assay (Pierce, Cat#23225) or similar.
Normalize samples to lowest protein concentration by adding lysis buffer.
Mix samples with 2x Laemmli sample buffer (62.5 millimolar (mM) Tris-HCl pH6.8, 100 millimolar (mM) DTT, bromophenole blue).
Heat samples for 00:05:00 at 95 °C
Samples are now ready for SDS-PAGE
5m
SDS-PAGE (Sodium dodecyl sulfate - polyacrylamide gel electrophoresis)
3h 30m
Use clean glass plates for casting gels in SDS-PAGE and assemble everything according to your set-up.
Cast resolving gel with percentage with appropriate percentage, i.e. 7.5 % for LRRK1 and LRRK2 analysis and 12.5 % for Rab GTPases.
Incubate for 00:40:00 to 01:00:00 at Room temperature to allow polymerization.
1h 40m
Cast stacking gel (5 % acrylamide) and insert comb.
Incubate for at least 00:30:00 at Room temperature to allow polymerization.
30m
You can also incubate the gel longer to ascertain the best possible polymerization.
Remove comb and clean pockets with Laemmli running buffer to remove remaining gel residue.
Fill device with Laemmli running buffer.
Load 8 µL protein marker (PageRuler™ Prestained Protein Ladder 10 to 180 kDaThermo Fisher ScientificCatalog #26616 or Spectra™ Multicolor High Range Protein LadderThermo FisherCatalog #26625 ), then load 25 µg per sample. Fill empty pockets with same volume of 2x Laemmli sample buffer as sample volume
Connect electrodes and run gel at 80V constant voltage for approx. 00:20:00 until samples have reached resolving gel.
20m
Run samples through resolving gel at 110 V constant voltage for approx. 01:00:00 until bromophenole blue has almost reached lower end of gel.
1h
Western Blot
5h 25m
Equilibrate gel from SDS-PAGE to Western Blot transfer buffer (12.5 millimolar (mM) Tris-HCl pH 8.3, 100 millimolar (mM) glycine) shortly
Build Western Blot sandwich by placing a 0.22 or 0.45 µm PVDF membrane (0.22 µm for proteins <50 kDa) on gel and surrounding by wet Whatman paper.
Avoid and remove air bubbles between gel and membrane to allow an error-free transfer.
Place sandwich in Western Blot chamber, fill with transfer buffer and start transfer at 60 V for 01:30:00 .
1h 30m
Check if all marker bands have been transferred onto membrane. If not, continue electrophoretic transfer for another 00:10:00 to 00:15:00 .
25m
Block the membrane to inhibit unspecific binding with 5 Mass / % volume milk in TNE-T buffer.
Incubate for 01:00:00 at Room temperature .
1h
Wash membrane multiple times with TNE-T buffer.
Add primary antibody in TNE-T (or 5 Mass / % volume BSA in TNE-T for phosphosite antibodies). Dilute antibody according to manufacturer instructions, commonly 1:1000 or 1:5000 in the respective buffer.
Incubate at 4 °C Overnight while shaking.
1h
Wash multiple times
Dilute secondary antibody, e.g. HRP-coupled anti-mouse or anti-rabbit antibody, in TNE-T 1:5000 and add onto membrane.
Incubate for 01:30:00 at Room temperature while shaking.
1h 30m
Wash membrane multiple times
Place membrane on tray and add detection reagent for HRP-based chemiluminescence detection so membrane is covered.
Place in LAS-3000 (Fujifilm) and detect chemiluminscence signal.
Take picture of marker bands on membrane.
Overlay picture to determine molecular weight of bands and if necessary, select and show bands-of-interest and adjust contrast and brightness to make signals better visible using ImageJ.
If necessary, strip membrane to detect using another primary antibody. Otherwise store membrane at 4 °C .
Strip membrane
10m
After detecting signals of phosphosites, it is necessary to strip membrane of antibodies to detect signal of respective proteins using another primary antibody.
Wash membrane in TNE-T once to remove detection reagent.
Add 10 mL stripping buffer (20 millimolar (mM) Tris-HCl pH 7.4, 6 Molarity (M) guanidine hydrochloride, 0.02 % (v/v) NP-40, 0.1 Molarity (M) β-mercapto ethanol).
Incubate membrane for 00:10:00 at Room temperature while shaking.
10m
Wash membrane in TNE-T and transfer to fresh box, and wash multiple times
Repeat steps 17-22 for new primary antibody detection.
Acknowledgements
This research was funded by Aligning Science
Across Parkinson’s (grant ASAP-000519 to S.R.P., S.K. and F.S) through the Michael J. Fox
Foundation for Parkinson’s Research (MJFF).