Jun 04, 2025

Western Blot

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyqknrvx9/v1
  • Lucas Blasco-Agell1,2,
  • Jara Montero-Muñoz1,2,
  • Meritxell Pons-Espinal1,2,
  • Antonella Consiglio1,2,
  • Miquel Vila3
  • 1Department of Pathology and Experimental Therapeutics, Bellvitge University Hospital- IDIBELL, 08908 Hospitalet de Llobregat, Spain;
  • 2Institute of Biomedicine of the University of Barcelona (IBUB), Carrer Baldiri Reixac 15-21, Barcelona 08028, Spain;
  • 3VHIR-CIBERNED-ASAP
  • Vilalab Public
Miquel Vila
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyqknrvx9/v1
Protocol Citation: Lucas Blasco-Agell, Jara Montero-Muñoz, Meritxell Pons-Espinal, Antonella Consiglio, Miquel Vila 2025. Western Blot. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyqknrvx9/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 04, 2025
Last Modified: June 04, 2025
Protocol  Integer ID: 219571
Keywords: western blot
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020505
Abstract
Western Blot
For protein isolation, cell pellets are resuspended in RIPA buffer with 1X protease inhibitors, sonicated, and centrifuged at 10,400xg for 10 minutes at 4°C.
Supernatant is collected and protein concentration is determined by Bradford assay.
Protein is mixed with 6x LSB (Sigma-Aldrich) and 4% DTT (Sigma-Aldrich) and heated at 95 ºC for 5 minutes in a Thermomixer.
Samples are subjected to SDS-PAGE on 8 to 12% of polyacrylamide and samples are run with a PowerPac Basic (Bio-Rad Laboratories) at 90-120 Volts for 60-90 minutes in a Mini-Protean‱ 3 Cell (Bio-Rad Laboratories).
Gel is transferred at 350 Amperes for 120 minutes onto pre-activated polyvinylidene difluoride (PVDF) or Nitrocellulose membranes in a Mini-Protean‱ 3 Cell (BioRad Laboratories). Correct protein transfer is checked by red Ponceau S Solution (Panreac).
Membrane is rinsed 3 times with TBS with 0.1% of Tween20 and blocked at RT for 1 hour with 5% milk or 5% BSA and incubated with primary antibody at 4°C ON.
The day after, membrane is rinsed and incubated with secondary Horseradish Peroxidase (HRP)-labelled antibody at RT for 1 hour.
After rinsing, signal is developed with ECL‱ start Western Blotting Detection Reagent (GE Healthcare Amersham‱) and images are obtained using a ChemiDoc System (Bio- Rad Laboratories).
Protein amount is expressed as a ratio between the band intensity of the protein of interest and the loading control protein β-Actin or Glyceraldehyde-3- Phosphate Dehydrogenase (GAPDH).