Protein extraction from frozen tissues?

lnd at mail.utexas.edu lnd at mail.utexas.edu
Fri Mar 26 12:01:06 EST 1999


I work with arabidopsis cells and to get total proteins (including membrane
and soluble ones) I usually freeze filtered cell in liquid N and make a fine
powder by grinding with mortar and pestle. I freeze them several time and
grind again. So finally the powder is very fine. Then all that material is
transferred into the tube containing 0.5 % SDS or other detergent containing
buffer with protease inhibitors (Complete Min, PMSF, EDTA, leupeptine..etc.)
and incubated at 4 C for 1 h  with continuing stirring.  If you are not
interested in native characteristics of target protein(s), you can add upto
2-8 % detergent and boil sample for 5-10 min.  Then use BCA assay to
determine protein concentration (you need to know how much proteins are you
loading onto gel), concentrate sample if necessary  and subject required
amount to SDS-PAGE with following transfer on NC or PVDF.

Hope it's helpful for you.

L. D.

UT @ Austin




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