How do you prepare a cell lysis buffer?

Question

Can you please explain step by step [detailed] from getting your cells out of an incubator. I'm a new intern and dont want to mess up. =] Thank you! Also, how do you go from there to performing a western blot. I know how to run it and transfer it just not sure how to get to that point. Also, it would be helpful if u told me how much (like how many micro liters of what to add to make the cell lysis buffer) Thank you!!!!

Answer 1

There isn't a short answer to your questions.  The type of cell lysis buffer you want depends on what you need: whole cell lysate, nuclear prep, immunoprecipitation, etc.

If you are just doing a standard western on whole-cell lysate, try laemmli buffer.  It's commercially available, and I believe it's relatively inexpensive.

Otherwise, I recommend checking the following forum for a suitable protocol:  http://www.protocol-online.org/

Good luck!

EDIT: do NOT follow nalaredneb's advice!  You need a lysis buffer that will inhibit protease activity.  If you merely scrape the cells up with a razor, the proteins will be degraded by proteases and your western blot will be useless.

Answer 2

Cells can be scooped up with a razor blade under the careful watch of a microscope.