Jan 09, 2025

Public workspaceWestern Blot - Frozen cell pellets

DOI
dx.doi.org/10.17504/protocols.io.ewov1d562vr2/v1
  • 1Laboratory of Molecular Neurogenetics, Department of Experimental Medical Science, Wallenberg Neuroscience Center and Lund Stem Cell Center, BMC A11, Lund University, 221 84 Lund, Sweden.
Laura Castilla-Vallmanya
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DOI: dx.doi.org/10.17504/protocols.io.ewov1d562vr2/v1
Protocol Citation: Anita Adami, Laura Castilla-Vallmanya 2025. Western Blot - Frozen cell pellets. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d562vr2/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: January 09, 2025
Last Modified: January 09, 2025
Protocol Integer ID: 117993
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research
Grant ID: ASAP-025170
Abstract
This protocol describes how to extract protein from frozen pellets and how to perform a western blot for protein detection.
Protein Extraction
Protein Extraction
1h
1h
Thaw the cell pellets TemperatureOn ice .

10m
Add RIPA buffer supplemented with protease inhibitors, vortex well and lyse the cells on ice for Duration00:30:00 .
(For a 200,000 cells pellet use Amount40 µL of lysis buffer, scale up or down accordingly)

30m
Centrifuge at Centrifigation12.000 rpm, 4°C for Duration00:20:00

20m
Carefully transfer the supernatant to a new tube. Store the protein lysate at Temperature-80 °C or continue with the steps in the Western Blot section.

Western Blot
Western Blot
6h 25m
6h 25m
Prepare gel samples:
  • Mix the appropriate amount of sample with 4x loading dye (LDS), 10x reducing agent and water.
  • Boil the samples for Duration00:05:00 at Temperature95 °C .
  • Spin briefly.


5m
Run the gel:
  • Load the boiled samples in a pre-made 4-12% Tris-glycine SDS-PAGE gel.
  • Load a well with a prestained ladder.
  • Run at 200V in 1x MES SDS running buffet for Duration00:45:00 at TemperatureRoom temperature .

45m
Transfer to membrane:
  • Transfer the proteins to a PVDF membrane using the Transblot-Turbo Transfer system (BioRad) following manufacturer's instructions.
  • Check the ladder has transferred well to the membrane.
10m
Blocking:
  • Cut the membrane if the size of the proteins to be detected is different enough.
  • Place the membrane in a cuvette making sure the protein side is facing up.
  • Briefly rinse the membrane with Tris-buffer saline with 0,1% Tween (TBST).
  • Block with 5% skimmed milk-TBST for Duration01:00:00 at TemperatureRoom temperature or DurationOvernight at Temperature4 °C .

2h
Antibody incubation:
  • Dilute primary antibody in 5% skimmed milk-TBST.
  • Incubate on a shaker DurationOvernight at Temperature4 °C . For abundant targets, incubate for Duration01:00:00 at TemperatureRoom temperature .

  • Briefly rinse the membrane with TBST.
  • Wash three times with TBST on a shaker for Duration00:10:00 at TemperatureRoom temperature .
  • Dilute species-appropriate HRP-conjugated secondary antibody in 5% skimmed milk-TBST.
  • Incubate on a shaker for Duration01:00:00 at TemperatureRoom temperature .
  • From this point, protect the membrane from light.

  • Briefly rinse the membrane with TBST.
  • Wash three times with TBST on a shaker for Duration00:10:00 at TemperatureRoom temperature .
  • Wash once with TBS on a shaker for Duration00:05:00 at TemperatureRoom temperature .

  • NOTE: If using an HRP-conjugated primary antibody, wash thrice and image (skip the secondary antibody step).

3h 25m
Detection:
  • Prepare the detection reagent following manufacturer's instructions.
  • Incubate on a shaker for following manufacturer's instructions.
  • Image with chemiluminescence using an appropriate imager.

Stripping:
  • Use a commercial stripping buffer as per instruction.
  • Briefly rinse the membrane with TBST.
  • Repeat blocking, antibody incubation and detection of the new target (usually the loading control).

NOTE: A large proportion of the protein will be lost during the stripping step. Always blot the most abundant target last.