Membrane protein extraction from tissue

Efficient extraction of cell membrane proteins are important for the sample preparation protocol and subsequent analysis by Western blot. When resuspending the pellet, used as a low volume of resuspension buffer as possible to obtain a sample of a high protein concentration.

Protein extraction process:

a. Start with 1 g of tissue washed in cold PBS

b. The use of liquid nitrogen to quick freeze the sample

c. Grind the tissue with a mortar

d. Place the fabric on the powder 10 ml lysis buffer

e. Using a Polytron, mix the soil and tissue lysis buffer (a blast - 20 seconds)

f. Centrifuge for 15 minutes at 500 x G at 4 degrees Celsius

g. Remove and save supernatant

h. Homogenize the pellet again with a further 5ml lysis buffer (20 seconds)

i. Centrifuge for 15 minutes at 500 x G at 4 degrees Celsius

j. Pool both supernatants

k. Centrifuge the supernatant at 45 000 x G for 15 minutes

l. Wash the precipitate twice in lysis buffer - the removal of the supernatant between washes

m. Resuspend in resuspension buffer




n. Store at -80 ° C, thaw for Western blot analysis (Aliquot to avoid multiple freeze thaw cycles)