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While we do not have a general protocol for tissue-based lysate preparation, here are some helpful tips:
Freshly prepared lysates result in fewer nonspecific and degradation bands, and therefore yield a cleaner blot. In general, tissue extracts tend to contain more background bands and degradation products than cell line extracts. Using fresh, sonicated, and clarified tissue extracts may lessen background. Samples should always be lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors. Please see our recommended inhibitors in the protocol below. We recommend the use of Cell Lysis Buffer #9803 for best results by IP.
Below is our in-house protocol for preparing tissue extracts for IP:
- Cut a small piece of tissue, approximately 3mm^3
- Place in a clean Dounce homogenizer
- Add 1mL cold 1X Cell Lysis Buffer #9803 + 1.0 mM