Hello everyone.
I'm trying to see secreted-phosphorylated-protein by western blot, but struggling with detecting it. There's nothing wrong with detecting whole-cell lysate samples but it's impossible for conditioned media samples.(It is secreting protein so exists in both.) No bands. Just BLANK. Even though I use same antibody for same time. I can detect control, TIMP2, so proteins are there...
I even used other lab mate's sample for positive control and suprisingly, it was vanished.
Can you help me?
I use conditioned media sample, which is incubated in OptiMEM for 48hrs and methanol/chloroform precipitated. It is 5X concentrated and boiled for 5min at 95°C.
I use 5% BSA for blocking and 1° antibody(PY1000).
Photos are my lysate membrane and CM membrane.
Thank you in advace!
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