Hi guys, it's my first post here. I've been reading you for years but have just decided to post now.

Im having some issues at looking for ALK ( ALK (31F12) Mouse mAb #3791) from Cell Signaling in some cell lysates that are ALK positive. They are lisated with RIPA + anti prot phosp combo.

I'm adding 40ug protein/well in a 10% gel (need to try others abs) for SDS page standard conditions. Electroforesis is running at 60V until the 2cnd gel, then 115-120V until finishes. Transfer with metanol and nitrocelulose 0,45 1h 100V surrounded with ice (we dont have a cool room)

Blocking with 5 % TBS BSA 1h RT, overnight primary AB with TBS BSA at manufacturers concentration (1:1000) and finally second AB for 1h at RT 1:2000. Washes are made with TBSTween finishing with a TBS one. Im using Biorad ECL (Clarity Max? 1:1) and exposing 8min

Im tryed other antibodies at the same membrane and worked well with this 40ug of protein, but literature and other images make me think that its a very low concentration to see ALK and P-ALK.

I have uploaded the ALK ab and the post GAPDH at the same membrane

Thanks so much,

Victor.