Sep 23, 2023
SDS-PAGE and western blot analysis
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Protocol Citation: Elias Adriaenssens 2023. SDS-PAGE and western blot analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj33plx9/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: June 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84147
Keywords: SDS-PAGE, western blot analysis, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes SDS-PAGE and western blot analysis.
Attachments
758-1926.pdf
47KB
Materials
Materials
- 100 mM DTT
- PageRuler Prestained protein marker (Thermo Fisher)
- nitrocellulose membranes (RPN132D, GE Healthcare)
- Mini Trans-Blot Cell (Bio-Rad)
- 0.1% Tween 20
- horseradish peroxidase (HRP)-coupled antibodies
RIPA buffer
| A | B | |
| Tris-HCl pH 8.0 | 50 mM | |
| NaCl | 150 mM | |
| sodium deoxycholate | 0.50% | |
| SDS | 0.10% | |
| NP-40 | 1% |
cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
Roche PhosSTOP™Merck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001
Pierce™ Detergent Compatible Bradford Assay KitThermo FisherCatalog #23246
SuperSignal™ West Femto Maximum Sensitivity SubstrateThermo FisherCatalog #34096
SDS-PAGE and western blot analysis
SDS-PAGE and western blot analysis
10m
10m
For SDS-PAGE and western blot analysis, collect cells by trypsinization and subsequent centrifugation at 300 x g, 4°C, 00:05:00 .
5m
Wash the cell pellets in PBS and centrifuge once more at 300 x g, 4°C, 00:05:00 .
5m
Remove the supernatant and lyse the cell pellets in RIPA buffer supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP, 4906837001, Roche).
After incubating in RIPA buffer for 00:20:00 On ice , clear the samples by centrifugation at 20000 x g, 4°C, 00:10:00 .
30m
Collect the soluble supernatant fraction and measure the protein concentrations using the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).
Then, adjust the samples for equal loading and mix it with 6x protein loading dye, supplemented with 100 mM DTT. Then, boil it for 00:05:00 at 95 °C .
5m
Load the samples on 4-12% SDS-PAGE gels (NP0321BOX, NP0322BOX, or NP0323BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).
Transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for 01:00:00 at 4 °C using the Mini Trans-Blot Cell (Bio-Rad).
1h
After the transfer, block the membranes with 5% milk powder dissolved in PBS-Tween (0.1% Tween 20) for 01:00:00 at Room temperature .
1h
Incubate the membranes Overnight at 4 °C with primary antibodies dissolved in the blocking buffer and wash.
1h
Wash for 00:05:00 .(1/3)
5m
Wash for 00:05:00 .(2/3)
5m
Wash for 00:05:00 .(3/3)
5m
Then, incubate with species-matched secondary horseradish peroxidase (HRP)-coupled antibodies diluted 1:10,000 in blocking buffer for 01:00:00 at Room temperature .
1h
Afterwards wash the membranes three times with PBS-T and further process for western blot detection.
Incubate the membranes with SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) and image it with a ChemiDoc MP system (Bio-Rad).
Analyze the images with ImageJ [57].