Homogenization and Solubilization of RASSLs
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1. Place 1 ml of homogenization buffer in a round bottom 14 ml tube.
2. Transfer the frozen tissue into the 14 ml tube. Homogenize using a homogenizer at max speed until tissue is fully ground (about 30-60 seconds).
3. Pipet the homogenate to an eppendorf tube and immediately place it on ice.
4. If the homogenate is too viscous, sonicate for 3-5 seconds.
5. Normalize samples by tissue weight and use approximately 10-50 mg of tissue for solublization.
6. Solublize each sample in an eppendorf tube using the following conditions:
* 1x SSB * 1x Triton-X 100 * 1x CompleteTM Cocktail (Protease Inhibitor, Boehringer #1697498)
Example:
x ml of homogenate (~10-50 mg tissue)