Protocol Citation: Alexandria White, Sean D. Kelly 2025. Western Blot Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwqpzv8j/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: December 19, 2024
Last Modified: January 30, 2025
Protocol Integer ID: 119308
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Against Parkinson's (ASAP)
Grant ID: ASAP-020527
Abstract
This protocol details the western blot analysis.
Image J Western Blot
Image J Western Blot
Drag Western Blot jpg file in Image J.
Click on ‘Image’ tab -> ‘Transform’ -> ‘Rotate’ and adjust image to make sure the lanes are horizontal.
Click on the ‘rectangle tool’ and select the bands you want to analyze.
Click on the ‘Analyze’ tab ->’Gels’ -> ‘Select First Lane’ (or Control 1) -> ‘Plot Lanes’ (or Control 3).
Click on ‘Straight’ tool to divide the peaks. Draw lines to divide peaks. Hold ‘Shift’ while drawing lines for a straight line.
Click on ‘Wand (tracing) tool’ and select each individual peak. A new window should pop up for the area under each peak.
Once you have all the areas (integrated densities) for each peak (band). Copy and paste the values into excel corresponding to their ID from the gel layout.
Do the same for the control protein.
Then divide the ID of your target protein/ID ctl.
Graph and do stats on these values in GraphPad.