Western Blot: Introduction to Blocking

Описание: Western Blot is a widely used research technique to detect proteins using antibodies. One important step of a western blot called ‘blocking’ prevents non-specific binding of antibodies to the membrane. Since the membranes used for a western blot have a high affinity for all proteins, the primary and secondary antibody may bind non-specifically to the membrane. Blocking covers up these non-specific binding sites, ensuring most of your primary antibody binds to the target protein.

The video lists the materials and reagents needed for blocking and how to conduct it. It is important to note that there are different blocking reagents which serve best under differing circumstances. Non-fat milk is cheap compared to BSA, readily available and easy to prepare. On the other hand, BSA provides a clearer result as it contains only one protein so there is less for the antibody to cross react with. It also works better with phospho-antibodies as albumin is a secreted protein and it tends to not be phosphorylated.

Additionally, you should also optimize the concentration of blocking reagents in your buffer. Too much blocking reagent may reduce the signal from your target protein.

This video was made by McMaster University students Aniqa Chaudhry, Brooke D'Mello, Ibrahim Ilahi, Kunwar Karim, Mubariz Maqsood, Adnan Moinuddin, and Julia Zhu in collaboration with the Demystifying Research McMaster 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.

This video is for general and educational purposes only.

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References:

Mahmood, Tahrin, and Ping-Chang Yang. “Western Blot: Technique, Theory, and Trouble Shooting.” North American Journal of Medical Sciences, Medknow Publications & Media Pvt Ltd, Sept. 2012, https://www.ncbi.nlm.nih.gov/pmc/arti....

Membrane Blocking for Western Blot | Sino Biological. https://www.sinobiological.com/catego....

Quantitative Western Blot - Science Topic - Researchgate.net. https://www.researchgate.net/topic/Qu....

Man, The Protein. “Western Blot Blocking: Tips and Tricks for Blocking Agents.” Western Blot Blocking: Tips and Tricks for Blocking Agents, https://info.gbiosciences.com/blog/we....