Removing fat from tissue preps?
seanpat at fmed2.uncu.edu.ar
Tue Jun 13 09:08:43 EST 2000
>Sean Patterson wrote:
>> Do you have a particular reason for thinking that lipids are the
>> problem? The slimy layer sounds like DNA to me.
>Whenever I've done protein preps before, the DNA has been a clear
>'gel'. This stuff is opaque and floats on the surface of the prep.
>Vigorous votexing/pipetting shears DNA and makes it go away - this stuff
>just diperses, then forms the same layer again. Plus, I'm getting the
>slimy stuff in the preps from tissue but not in the cell preps.
Okay - sounds like lipid.....
I do simultaneous analysis of lipids and proteins in cell preps and
subcellular fractions, for instance synaptosomes, but not tissue preps.
However, it should be easy to adapt the method once you have everything in
solution/suspension. Just add 10 volumes of chloroform/methanol (1:1), mix
well (I vortex for one minute then give it five minutes in a bath
sonicator) and spin out the precipitated proteins in a microfuge (5 min at
max). The one-phase supernatant will have virtually all your neutral lipids
and most of the phospholipids - and only very hydrophobic proteins (e.g.
some myelin proteins are quantitatively extracted into the supernatant).
The only fiddly bit is drying the pellet before going into sample
buffer - if you over-dry it can be a pain getting the proteins back into
solution. I usully just leave the tubes open on the (well-ventilated) bench
until the olfactory test fails to detect any chloroform - and before anyone
posts, yes I know it's not healthy to sniff chlorinated organics. I usually
stick to ether.
This will, of course, also remove any detergent you used in the
homogenization step, but almost any organic extraction that removes lipids
will do the same.
Hope this is of some use.
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