Hi all
I´m trying to quantify pAkt levels in rat gastrocnemius muscle. I´m using the antibody dilutions proposed by the manufacturer (Santa cruz, 1:1000), and I´m getting very "weak" and unspecific (there appear also other bands) bands. I use the secondary rabbit antibody in a 1:5000 dilution. With the Akt antibody (SC 5298) I have no problems (strong and specific bands).
I don´t know if anyone ever had similar problems. I would really appreciate any suggestion/advice.
Thanks for reading.
Iñaki Milton
What is your dilution with SC antibody? pAKT at Ser473 from SC will work much better for IHC than western analysis. Use cell signaling antibody that works better than SC for Western blot analysis. If you can't afford, try same SC antibody at 1:500 dilution in 1% milk/casein TBS-T buffer over night at 4oC.
We normally use SC antibodies at 1/250 for western blotting. For phospho blots make sure you have phosphatase inhibitors in your lysis buffer and use TBS rather than PBS for your wash buffer. BSA is also better for blocking rather than milk which can contain phospho proteins can could be increasing your background signal.
Hi Inaki. You can find alot of information about antibodies against pAKT on www.pAbmAbs.com. This is a website where scientists can share information about the antibodies they use in their research. i also hope you guys will share your experience with the antibodies you use in your research!
Kind regards,
Simon
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