Western Blot: Result Interpretation

The western blot is a widely used research technique that detects proteins using antibodies. One important step of a western blot is the analysis and interpretation of collected results.  Many aspects must be considered when analyzing a western blot, including how different immuno-blotting techniques may affect your results depending on their individual strengths and limitations. This video lists three popular options for immuno-blotting techniques: colorimetric or chromogenic, chemiluminescent, and fluorescent detection. In addition, techniques pertaining to band analysis, quantification, and normalization are discussed in regards to the analysis of western blot results.

The video includes important factors and steps to consider when analyzing the results of your western blot using footage from a laboratory at The Research Institute of St. Joe's Hamilton/McMaster University and diagrams created using BioRender.

This video was created by Brooke D’Mello, Ibrahim Ilahi, Adnan Moinuddin, and Julia Zhu in collaboration with the McMaster Demystifying Research Program.

We would like to also acknowledge the contributions of Dr. Takuma Isshiki, who provided guidance in the process. Finally, we would like to also thank Dr. Kjetil Ask and the Ask Lab for the additional guidance, insights and support.

Episode #1: Introduction to Western Blotting: Introduction to the Western Blot

Episode #2: SDS-PAGE: Western Blot: Introduction to SDS-PAGE

Episode #3: Wet Transfer: Western Blot: How to do a Wet Transfer

Episode #4: Electroblotting: Western Blot: Introduction to Electro...

Episode #5: Blocking: Western Blot: Introduction to Blocking

Episode #6: Immunodetection: Western Blot: Introduction to Immunod...

This video is for general and educational purposes only.

Subscribe to the Demystifying Research McMaster channel.  

Copyright McMaster University 2022

References:

Bass, J., Wilkinson, D., Rankin, D., Phillips, B., Szewczyk, N., Smith, K., & Atherton, P. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scandinavian Journal of Medicine & Science in Sports, 27(1), 4–25. https://doi.org/10.1111/sms.12702

Detection Methods. (n.d.). Bio-Rad Laboratories. Retrieved August 23, 2022, from https://www.bio-rad.com/en-us/applica...

Fluorescent Western Blotting. (n.d.). Bio-Rad Laboratories. Retrieved August 23, 2022, from https://www.bio-rad.com/en-us/categor...

Ghosh, R., Gilda, J. E., & Gomes, A. V. (2014). The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Review of Proteomics, 11(5), 549–560. https://doi.org/10.1586/14789450.2014...

Image Analysis and Quantitation for Western Blotting. (n.d.). Bio-Rad Laboratories. Retrieved August 23, 2022, from https://www.bio-rad.com/en-us/applica...

Sawyer, A. (2018, September 19). Choosing the right detection method for your western blot. BioTechniques. https://www.biotechniques.com/news/ch...