12 December 2017 4 8K Report

Most protocols suggest using a lysis buffer, spinning, and then adding Laemmli buffer to the supernatant. However, I have heard that it is easier to simply lyse the cells with 1x Laemmli buffer, heat at 100C for 10 minutes, quench on ice, quick spin, then load. Is this a better protocol for westerns? It certainly seems easier. Apparently, this method prevents the loss of nuclear/golgi proteins because the lysate is not spun at a high speed. The only problem seems that this protocol can produce a very sticky lysate that is hard to load. I have heard that this is due to lipids.

What is your lysis protocol for a western blot? Why must lysis buffer be used on ice? Are nuclear/golgi proteins lost in the pellet during high speed spins?

Thanks!

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