Feb 29, 2024

Public workspaceWestern blot for tissue extract

DOI
dx.doi.org/10.17504/protocols.io.yxmvm38q9l3p/v1
  • 1KU Leuven. Neurobiology and Gene therapy lab
María Sanchiz Calvo
Open access
DOI: dx.doi.org/10.17504/protocols.io.yxmvm38q9l3p/v1
Protocol Citation: María Sanchiz Calvo, Eduard Bentea, Veerle Baekelandt 2024. Western blot for tissue extract. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm38q9l3p/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: February 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95960
Keywords: ASAPCRN
Abstract
Protocol for the detection of proteins by Western blot from tissue extract
Materials
Recipe for 100 mL 1x RIPA buffer :
o 5 mL Tris pH 7.4
o 1 mL Triton x100
o 1 g Deoxycholate
o 3 mL 5M NaCl
o 200 µL 0.5M EDTA (pH 8.0)
o 1 mL 10% SDS
Add water until 100 mL (MilliQ water)
Add protease and phosphatase inhibitors (Pi + PPi) right before use
Reagents used during Western blotting :
o 4X Laemmli buffer
o PageRuler‱ Plus Prestained Protein Ladder
o 4–15% Criterion‱ Tris-HCl Protein Gel
o Trans-Blot‱ TurboTM PVDF Membrane
o 5% milk powder dissolved in PBS-T 0.1% (PBS + 0.1% Triton-X100)
o Goat anti-rabbit HRP secondary antibody (Dako)
o ECL Prime chemiluminescence kit (GE Healthcare)
Recipe for 10.2 mL 4x Laemmli buffer (0.24M Tris pH 6.8, 7.27% SDS, 40% Glycerol, 10% b-Mercaptoetanol, 0.01% Bromophenol blue):
  • 2.4 ml 1M Tris pH6.8
  • 0.8g SDS
  • 4ml 100% glycerol
  • 2.8ml dH2O
  • 1ml b-mercaptoethanol
  • 0.01% Bromophenol blue
Before start
Perform protein extraction from snap-frozen brain tissue: - weigh tissue - add RIPA buffer (see Materials) : 10 X of the weight (40 mg = 400 µL) - homogenize samples using sample homogenizer - sonicate samples at 4 degrees C, 3 times 15 seconds (keep the samples on ice between each sonication) - centrifuge samples at 6000 g for 10 minutes at 4 degrees C - collect supernatant and measure protein concentration - aliquot protein extracts and store at -20 degrees
Day 1
Day 1
Prepare sample for western blot
Amount15 µg of protein in Amount12 µL PBS
Add Amount4 µL Laemmli buffer
Boil at Temperature98 °C for Duration00:10:00

10m
Load samples, together with Amount7 µL of mass marker (PageRuler‱ Plus Prestained Protein Ladder) on a on 4–15% Criterion‱ Tris-HCl Protein Gel.

Run the gel for Duration00:10:00 at 80V

10m
Run the gel for Duration00:45:00 at 150V

45m
Transfer the proteins on PVDF membrane (Trans-Blot‱ TurboTM PVDF Membrane) using Trans-Blot Turbo Transfer System (Bio-Rad), using the pre-programmed protocol STANDARD SD: Duration00:30:00 , up to 1.0 A, 25 V.

30m
Block membranes for Duration01:00:00 using 5% milk dissolved in PBS-T 0,1% at TemperatureRoom temperature

1h
Incubate membranes DurationOvernight in primary antibody in 5% milk PBS-T 0,1% at Temperature4 °C

Day 2
Day 2
Wash membranes with PBS-T 0,1% for 10 minutes, 3 times at TTemperatureRoom temperature
Wash PBS-T Duration00:10:00
Wash PBS-T Duration00:10:00
Wash PBS-T Duration00:10:00
30m
Incubate membranes for Duration01:00:00 horseradish peroxidase-conjugated secondary antibody (Dako) diluted 1/10000 in 5% milk PBS-T 0,1% at TemperatureRoom temperature

1h
Wash membranes with PBS-T 0,1% for 10 minutes, 3 times at TTemperatureRoom temperature
Wash PBS-T Duration00:10:00
Wash PBS-T Duration00:10:00
Wash PBS-T Duration00:10:00
30m
Develop membranes using ECL Prime (GE Healthcare)