Tissue samples should be collected, weighed, and added to lysis buffer (100 mg of tissue per 900 μL 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 nitrogen 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 1 μg/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 2-8 °C. Following homogenization, the tissue preparation is centrifuged for 2 minutes in a microfuge at 13,000 xg. Making sure that the cell pellet is not disturbed; aspirate the supernatant. The sample must be frozen immediately and if stored, placed in a -80°C freezer. When you are ready to ship the samples, they must be placed in dry ice and shipped by an overnight carrier.