Protein Lysate Preparation Protocol (Tissue)
Scott Vermilyea
Published: 2024-03-21 DOI: 10.17504/protocols.io.n2bvj3k3xlk5/v1
Abstract
This protocol details the preparation of protein lysate of the tissue sample.
Attachments
Steps
Protein Lysate Preparation (Tissue)
1.
If tissue stored at -80°C, place in -20°C for at least 4h 0m 0s or prior to homogenization.
2.
Weigh tissue out in mg.
3.
Add in 10 volumes of 1X TNE (e.g. 10µL of TNE per 1mgof tissue).
4.
Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and On ice.
5.
This is TNE crude lysate (no detergents).
6.
Add equal volume of 1xTNE +1% SDS, 0.5% NP-40, and 0.5% Doc (Complete TNE).
7.
Sonicate at 40°C (3 pulses: 10s On/2s Off at 20% amplitude).
8. STOPPING Point: Sample should be in “Complete TNE”. Make sure to sonicate and boil prior storage.
Boil (0h 10m 0s).
Note
9.
Centrifuge (16000x g,4°C).
10.
Collect supernatant for BCA protein assay.
