TISSUE HOMOGENATES PROCEDURE

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.

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