Do you collect tumor lysate for WesternBlot or Elisa? In my case, I use NP40 buffer + Proteinase Inihibitor cocktail -> homogenize tumor mass -> on ice for at least 30 min -> cold centrifuge -> collect supernatant.

Actually is for cytometry and ELISA. Thanks :)

Elisa would be ok but not for FACS! FACS needs intact cells. So we may need a protocols for single cell isolation.

For single cells isolation: collect tumor -> cut into small pieces -> add Trypsin 0.25% -> shacking in 4oC for 2h -> transfer to 37oC incubator 20 min -> through a strainer 100um -> centrifuge -> add ACK buffer (if needed) to lyse red blood cells -> centrifuge -> add FACS buffer -> count cell numbers -> go for staining -> FACS reading

 Hi Laura,,

You can use this:

Starting material: Tissues stored as chucks under liquid nitrogen.

During handling the material is kept on ice. Before generating a lysate, the tissue is first cut into ~1mm cubes by using a razor blade on a glass plate held on ice.

The small cubes are then transferred into a hand held potter homogenizer with three ml ice-cold RIPA buffer per gram of tissue.

 Tough tissues like Prostate, Skin and Thyroid need incubated in the RIPA for 20

min on ice before starting homogenizing

 Medium tough tissues like Colon, Duodenum, Heart, Kidney, Skeletal Muscle or Tonsil, are kept in RIPA on ice for 10min before homogenizing

 Homogenize by pushing the piston slowly into the mix by continuously twisting the wrist thus turning the piston around its axe.

 Make sure all tissue chunks slide between the piston and the glass wall while homogenizing.

 Once the piston reaches the bottom, reverse the handling

 Keep the tissue submerged in the ice during the process

 Repeat the cycle until the tissue is liquefied

 Divide the liquefied tissues over 1.5ml tubes and centrifuge at 13,000rpm for 3 min at 4C

 Transfer the clear supernatant in new clearly labelled tubes. Take 20ul out for protein determination

 Protein determination is carried out using the BioRad Protein Determination Kit.

 Adjust the lysate to 5mg/ml by adding ice cold RIPA buffer

 Store in liquid nitrogen.

RIPA buffer: 20mM Tris-HCL pH7.4, 150mM Na\Cl, 1mM EDTA, 1% Triton-X100, 1%

sodium deoxycholate, 0.1% SDS with freshly added PMSF to 1mM and with freshly

added aprotinin and leupeptin to 5ug/ml just before use.

All the best.

Hi, can anyone share protocol for preparing tumor lysate for Western blot? Thanks.