Tissue samples should be collected, weighed, and added to 9X volume of lysis buffer. Our recommended lysis buffer is 50 mM Tris-HCL with 2 mM EDTA, pH 7.4. If the samples are not homogenized immediately then the samples should be frozen in liquid N2 and stored at -80° C. While EDTA is a good inhibitor of divalent metal requiring proteases, you may want to minimize other protease activity by adding the following inhibitors: aprotinin, antipain, leupeptin, and pepstatin A (all at 1ug/ml) and 2mM PMSF (phenylmethylsulfonyl flouride).
Tissues may be homogenized using a Potter-Elvehjem homogenizer (Teflon pestle and glass mortar) attached to a variable-speed drill, a polytron or a tissuemizer. During the homogenization process, the tube should be submersed in an ice bath to maintain the sample at 4° C. Following homogenization, the tissue preparation is centrifuged for 2 minutes in a microfuge at 13,000xg. Making sure that the cell pellet is not disturbed; aspirate a minimum of 240 µL per sample to be shipped to us. The sample must be frozen immediately and if stored, placed in a -80° freezer. When you are ready to ship the samples, they must be place in dry ice and shipped by an overnight carrier.