Why lyse on a plate and scrape?

[hagereseb]

I have always wondered why when lysing cells for western blot (or other applications) people add the lysis buffer and then scrape. why not trypsinise cells and then wash and lyse them on a tube. i can understand why it might be important to avoid trypsin for cell membrane proteins. can anyone tell me if i can actually detach cells by trypsin, wash the heck out of the cells (with PBS) and then lyse in a tube.

[admin]

Hey there welcome to the forums , usually trypsin is not used as it is a protein (can mess up a lot of experiments) and there is an easier method call passive lysis if you find it hard to lyse (or just time consuming). I am not sure there are other reasons my mind is drawing a blank now.

[Clouseau]

I used to look at rapid phosphorylation events, so I had to perform quick lysis by scraping cells in the presence of boiling-hot (!) buffer. Nowadays I'm working with a different protein, so I routinely trypsinize cells, wash with PBS, and lyse in tubes. Works great!

[youbati]

Hi there,

I can confirm that trypsinizing, PBS-washing cells before lysis in tubes fairly works. Plus, with some experience, you may adjust the volume of lysis buffer to get less or more concentrated samples, which you cannot by lysing cells on plate, I mean if you lyse on plate, you can dilute your samples but never concentrate.

I usually perform at least 2 PBS washing steps, to get rid of all the trypsin. I harvest my cells in 15 ml Falcon tubes, make one wash in 5-6 ml PBS, then I transfer to eppendorf tubes, and make another wash in ~1 ml PBS and sometimes a third one in 400-500 µl.

For each step, I remove supernatants with a vacuum pump equipped with a fine gel loading-specific tip. That represents additionnal work, but it is worth.