Mouse Fibroblasts - need to extract nuclar protein (Nov/01/2009 )

Hi there,

I just began my bachelor thesis and have to extract a nuclear protein from mouse fibroblasts (to perform a Western Blot afterwards). Is it a good idea to use mouse ear tissue or do you have any better ideas? Maybe you have experience with that kind of experiment?!

Thanks in advance!
Isi

-Isi85-

You want some sort of actively dividing tissue that will produce fibroblasts, skin will work but is prone to contaminations of yeast and bacteria... embryos are the best and easiest to make sterile from the start.

Have a read of Freshney's Culture of Animal Cells, a laboratory manual for some good tips and techniques.

-bob1-

Thanks for your reply. But I think that is a misunderstanding. I don't want to establish a cell culture of MEF, but rather want to extract a nuclear protein from mouse ear cells.

Thank you!

-Isi85-

Ah, OK, you are aware that the ear is largely not composed of fibroblasts? In fact there will be very few even if you use a whole ear.

-bob1-

Isi85 on Nov 1 2009, 09:48 AM said:

Yep, any connective tissue will due. As it is a nuclear protein use loading buffer for lysis, and adding deoxycholic acid may help. Use a harsh method such as rotor-stator or bead beating.

-Feelcontraire-