(by bzahedi from bccrc.ca)
Thu Aug 19 13:28:06 EST 2010
Hi Chris, i've never looked at cardiamyocytes. But i do westerns for pAKT all the time and i feel your pain! But i have gotten them to work routinely.
i don't have a lot of time today so i'll answer you quickly and email me if you need more info.
-you need at least over 10 and most likely over 20ug of protein loaded to see a good signal. It is great if you are stimulating your cells because then you can tell if your problem is low abundance of pAKTgenerated by your system vs. due to your western conditions. So if you stimulate (activate) a cell surface receptor upstream of AKT and compare nil to stimulated, you should see an increase in pAKT.
- you can always use Jurktat Ts as a control as well, their pAKT levels are high.
- i am currently using the pAKT ab from Invitrogen because the last few batches of the Cell Signalling ab have been crap. if you are using this ab and have bought it in the past few months, try to get your money back. a bunch of us did. If you are using the older version of the Cell signalling ab ( ie they now have a new set of Abs they sell). try using it as a 1/500 overnight in 2% BSA.
-If you are using non fat milk, be carefull, the stuff from the store has phosphatases in it and wipes out the pakt signal. even though they don't recommend it, most of us at my work that are successfull with pakt staining where it is weak use bsa. you can always order phosphatase free nonfat milk. someone sells it, not sure who.
- Also the Cell signalling should send you a aliquot of their new ab. it works nicely, but you can only use it once ( we usually reuse our Abs and keep them in azide at 4 degrees for months at a time-you have to test this for each specific Ab though). Do not try to use it more than once if your pAKT levels are low in your system because you will be cursing and disappointed!
- what i do for my western protocol:
block membrane n 5% bsa for 20-30 min no more
add 1/1000 Invitrogen pAKT ( i can't remember the code but it says its for 10 reactions on the website incase you are looking it up) in 2%BSA+ Azide+TBST put at 4degreesC overnight- RT doesn't work well.
Wash next day with TBST 10minX3.
Add secondary as you usually would at RT for 1 hour.
wash same as before and detect.
I sometimes get a non specific band which is MUCH more visible than my pAKT which will be the weak stuff below this band. This could be only in my cells though as some of my friends who work with other cell types don't get this.
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] On Behalf Of Chris McDermott-Roe [cjmcdermottroe from googlemail.com]
Sent: August 19, 2010 5:38 AM
To: methods from magpie.bio.indiana.edu
Subject: phospho-Akt blotting
Can somebody provide me with a robust blotting protocol for phospho-Akt (in
cardiacmyocytes preferably)? If you could tell me which Ab and blotting
conditions work best, that would be really excellent!! Safe to say I'm
tearing my hair out in frustration.
thanks as always
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