Been searching for protocol online that deals with extraction of proteins from the mouse liver, heart, kidney and brain. Many protocol look as simple as extraction from adherent cell lines (i.e. add lysis buffer, homogenise and spin down).
What are the tips and hints that needs to be aware of for a first timer?
I heard about the need for an ultracentrifuge to spin down tissue lysates (to separate lipids and other debris??)
I'm also aware that some of these organs are drenched with blood cells (liver, kidney and heart). Need a special step to rid off blood cells?
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bob1
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Posted 20 August 2012 - 02:43 PM
Well, they are just composed of cells after all...
Add lots of protease inhibitors for liver. You don't need an ultrafuge for this, so long as you aren't working with particularly fatty tissue (e.g. brain), you shouldn't get too much lipid coming through. The lipid will also float - so can be removed by pipetting to a large extent.
You can perfuse the mouse with saline or PBS before sacrificing if you desperately need to get rid of the blood cells.
Add lots of protease inhibitors for liver. You don't need an ultrafuge for this, so long as you aren't working with particularly fatty tissue (e.g. brain), you shouldn't get too much lipid coming through. The lipid will also float - so can be removed by pipetting to a large extent.
You can perfuse the mouse with saline or PBS before sacrificing if you desperately need to get rid of the blood cells.
