what happens if you centrifuge lysed cells for too long?

Question

well i was trying to isolate cytosolic protein, and the protocol called for 5mins at 14000 rpm, i may have gone 7-12 minutes at that rate. There was a gelly look to the supernatant that i wound up collecting.

Answer 1

It doesn't sound like you're doing a strict protocol. A few minutes more should not matter, it may even have helped sediment more material into the pellet.

I say that this isn't a strict protocol because when making S100 (cytosolic) extract, one would typically spin at 100000 x g for over an hour. Doing a quick spin in a tabletop microcentrifuge would give a crude supernatant with many contaminating proteins.

Answer 2

Everything goes to the pellet, that's what happens. You need to centrifuge just enough to separate the layer and avoid pelleting everything.

Answer 3

Cells that are lysed have already spilled their contents. Therefore you would essentially be blending these contents into a mass.

Answer 4

too long, nothing really. you may separate materials that you didn't intend to.