Weak Western band for phosphorylated Akt - whats happening?? (Oct/26/2005 )

Hi Pria,

Just a thought but given your differences in yields between your sonication and tissue lysis, is your Bradford assay compatible to the detergent in the lysis buffer??????? This may result in you getting a false higher reading and your actual protein concentration may be much lower.

Just another idea on the pile.

Cheers,

Scott

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Thanks a lot guys for all the input:

First Scott..The Bradford assay is not the issue. Coz the sonicationa dn lysis were done in the same buffer. Also I use 2ul of the sample in 1 ml of the bradford dye....

Now to mickihiiri...
They were cells from the same batch though one was plated a week before the other...

I make 4 lts of transfer buffer....methanol and all at one time and use it till I finish it say 4 times. I dono t reuse used buffer though. Also never had problems with any other protein. I think I should start adding methnol just on the day of the western. My blots are in the secondary right now. Since I made the transfer buffer fresh yesterday..lets see what happens. I am really hoping that it is the transfer buffer issue.

You say 5 misn is long ?? hon...I have to expose my film for 2 h when my P-AKt bands are light.

Cell signaling have promised to help me. They said they would send me some positive cells so lets see...

My total Akt bands are never a problem even when my p-akt band is very light...knock on wood..

I transfer at 100 V for 1h (never been good at the mamps thing)

I load 50 mgs of protein/well.

I really appreciate all your input and please let me knnow if you think of something however, trivial. I thing i will go crazy if i dont figure it out. I will let u know how my blot develops today and I REALLY hope its the transfer buffer.

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UPDATE GUYS!!!!


Looks like the transfer buffer is the issue...my blot came out fine...a min exposure...
Planning to redo it and confirm for sure...

Thanks guys..appreciate all ur help.

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Bad news :-(
looks like the transfer buffer is not the issue. I think it might be the cell density of the plate...thats the only thing left or its a jinx.

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DOn't ever follow brochures blindly, I know it sounds weird but it might be a typo that they wrote blocking in milk, phospho-Ab will never work in milk, jsut block in BSA and I believe your prob will be solved!

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DOn't ever follow brochures blindly, I know it sounds weird but it might be a typo that they wrote blocking in milk, phospho-Ab will never work in milk, jsut block in BSA and I believe your prob will be solved!
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I agree with Smoochie. Pria, if you are still having problems I strongly suggest you try 5% BSA for blocking and antibody dilution if you haven't already. The lab I got the protocol from that I gave you awhile ago use the P-Akt antibody extensively, and all the advice I've gotten from them in the past has been dead on. I've also read in other posts that milk can have phosphatases in it which, may pose a problem for using phospho antibodies.

Good Luck,
Mountainman

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The problem is milk has phosphates in it. Remember that part of the epitope that the antibody recognizes is a phosphate group, so having any phosphates in the western buffers is a big no no when using phospho specific antibodies. Definitely block using BSA or gelatin.

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Just did two blots probed with pAKT from cell signaling. Used milk for blcoking, antibody 1:1000 in 3% BSA and the result is great. The same antibody worked for neighbour labs beautifully too.

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