brain protein isolation for WIB - can I simply use 4xLB+BME? (Aug/11/2005 )
I am looking to do an immunoblot for glutamate receptors and need to get cerebellum tissue as a positive control. Can I simply homogenize the tissue, spin, and add the supernatant to loading buffer with BME or do I need to use a different lysis buffer? Any advice is appreciated !
When you say homogenize the tissue, do you mean in H2O? Another buffer?
In general, a suitable buffer would be beneficial for the homogenization, it should include the following:
- A pH-buffering component (phosphate, tris etc.), pH ~ physiological
- ionic strengt (e.g. 150mM NaCl)
- detergents: Your protein is a membrane bound receptor and thus is highly hydrophobic and will probably solve poorly in water. Add e.g. 1% Triton X-100 (for membrane protein solubilization) and 1% SDS (protein interaction disruption and denaturation)
- protease inhibitors to prevent proteolysis.
Then spin, discard the pellet, and load the supernatant with loading buffer onto gel .
That's a big help, I don't normally do receptor stuff so this is all kinda new! I'll give it a try.