Mar 01, 2025
Version 2
Western Blot V.2
- 1Caltech
Protocol Citation: Chelsie Steele, Yujie Fan 2025. Western Blot . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk96p6v5r/v2Version created by Yujie Fan
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 16, 2025
Last Modified: March 01, 2025
Protocol Integer ID: 123609
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
Western Blot using the iBind Automated Western Systems
Materials
Collecting cells from the plate
Collecting cells from the plate
15m
15m
This protocol is written for a 6-well plate, please adjust the medium/buffer volume based on plate size.
Aspirate the medium in the wells and wash once with pre-cold DPBS. Add 1 mL of cold DPBS to each well, and collect the cells with cell lifter.
10m
Centrifuge the tubes at 300g for 5 minutes. Discard the supernatant. If the WB is not performed immediately, the pellet can be stored at -80°C.
5m
Preparing Western Blot lysate with RIPA buffer
Preparing Western Blot lysate with RIPA buffer
55m
55m
Prepare 1x RIPA buffer to lysis the cells. Use 10x RIPA buffer (Millipore 20-188) to prepare 1X RIPA buffer (2mL RIPA buffer + 18mL H2O).
5m
Add protease inhibitor cocktail (1x RIPA buffer + Protease inhibitor cocktail (1:100). At this step, 1x RIPA buffer with inhibitor can be aliquoted and frozen down at -20°C.
5m
Add 100 ul RIPA buffer to the cell pellet and re-suspend the cells. Leave the RIPA lysate on ice for 30 minutes. Pipetting to mix every 10 minutes until the lysate is clear.
30m
Centrifuge the lysate for 10 minutes at 15000g at 4°C (important that it is done at 4°C). Transfer the supernatant to a new 1.5mL Eppendorf tube. Make sure that the step is performed on ice. The supernatant will be your samples for the Western Blot.
15m
At this step, cell lysis can be stored at -80°C for long-term storage.
Measuring Protein concentration Using Qubit Protein BR Assay
Measuring Protein concentration Using Qubit Protein BR Assay
40m
40m
Use the Qubit Protein BR Assay kit (A50668) and Qubit4 to measure the protein concentration. In the 0.5mL Qubit Invitrogen tubes, prepare a Blank, Standard 1, Standard 2, and samples according to the following, ensuring that the order added is Sample (or Standard),, followed by the BR buffer, followed by the kit reagent:
Blank: Add 170uL of BR buffer + 30uL of Reagent
Standard 1: Add 20uL of S1 + 150uL BR Buffer + 30uL Reagent
Standard 2: Add 20uL of S2 + 150uL BR Buffer + 30uL Reagent
Sample: Add 2uL of Sample + 168uL of BR Buffer + 30uL of Reagant
20m
Vortex each of the blank, standards, and samples, and leave at room temperature for 10 minutes. Measure the protein concentration.
20m
Sample Preparation and Boiling
Sample Preparation and Boiling
5m
5m
In a separate 1.5mL Eppendorf tube, prepare a 1:9 ratio of 4x Laemmli Sample Buffer to 2-mercaptoethanol. Prepare enough for 34uL of this mix per sample.
Add 34uL of the loading mix to 100uL of lysis sample, and boil the samples for 3-5 minutes. At this point, prior to boiling, samples can be stored long-term at -80°C.
5m
Gel Preparation and Running
Gel Preparation and Running
1h 20m
1h 20m
After boiling samples, prepare 1X running buffer: mix 100mL of 10X Tris/Glycine/SDS Buffer with 900mL of Milli-Q water.
5m
Prepare mini-protein TGX stain-free gels (4-20%), in the BIO-RAD running cassette, and add running buffer.
5m
Load samples onto wells depending on protein concentration (up to 20uL/well). Add 8uL of loading marker dye to each end.
10m
Run the gel at 100V for 10 minutes, and then at 150V for 30-40 minutes.
1h
Transfer with Trans-Blot Turbo Transfer System
Transfer with Trans-Blot Turbo Transfer System
1h 30m
1h 30m
Transfer gel to PVDF membrane using the Trans-Blot Turbo Mini 0.2 um PVDF Transfer Packs. Transfer for 30 minutes.
30m
After the transfer, membranes are placed in iBind buffer. If alpha-synuclein and pS129-alpha-synuclein are to be blotted, fix the membrane with 4% PFA in PBS for 10 minutes and wash with PBS for 30 minutes before blotting.
1h
Protein Immunodetection with iBind Automated Western Systems
Protein Immunodetection with iBind Automated Western Systems
8h 40m
8h 40m
Following transfer, prepare 1X iBind Solution by adding: 300uL of 100X Additive + 6mL iBind 5X buffer + 23.7mL H2O to a 50mL falcon tube.
10m
Prepare primary antibodies, secondary antibodies and washing buffer according to iBind kit protocol. Add them to iBind Automated Western Systems and run overnight.
8h
Detect the signal with Max Western ECL and image with Biorad ChemiDoc Imaging system
30m