I wonder if anybody has experience with 3D cell aggregate model in drug development. I grow the aggregate and treat with specific drug (EGFR inhibitor), then get protein extraction and run SDS-page gel. The bands of p-EGFR with different concentration of the EGFR inhibitor looks the same. I wonder if anybody else has done the same experiment.
There are a few questions i have to ask you in this situation;
1. Do you detect a band around 180KDa or are you detecting some none specific band?
2. Do you use a house keeping antibody ie GAPDH, Actin, to show you have equal loading?
3. Have tried different blocking agents ie BSA, Mavel Milk, Fish skin gelatin?
4. Do you have a positive control on your blot ie HELA HepG2?
5. Did you optimize your antibody concentration and protein loading?
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