Hi everyone !
I have a protein of interest that "disappears" following combination of Starvation then SERUM refeed.
Here is the protocol I follow : I starve my macrophages cells OVN in RPMI without serum. After starvation, cells are put in rich media containing 20% FBS+human_serumAB. My read-out is WB: I extract cell lysates before the starvation, right after starvation (but before serum addition), and at various time point following serum refeed then I proceed for WB.
Before and after starvation, I can see the band corresponding of my protein.
However this band disappears very quickly (15min) after serum addition.
Blocking the proteasome with MG152 does not reverse this.
Blocking the lysosome functions using Chloroquine or Bafilo A1 does not reverse this neither.
Blocking macroautophagy specifically does not change the results. Nor mTOR blockade (since mTOR is involved in nutriments sensing).
I used various polyclonal antibodies with the same result. So this seems to exclude post-translational modification such as phospho, that could have modify the epitope for one monoclonal antibody, but not for numerous polyclonal.
One possibility would be induced secretion in the media but in 15min the timing seems short. I am doing a test on ultracentrifugated SN...update coming.
Does someone has an idea / hypothesis ?
Thanks !
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs