7 replies to this topic

[baboon]

Hi

I am trying to probe my western blots with P-akt (Thr 308) and P-akt (Ser 407) abs from cell signaling. I have repeated the western  2 times but I still don't get any bands. Wonder where am I going wrong?
Could it be the dilutions of the primary and secondary antibodies that I am using?
I am loading 40ug of protein and tried using dilution (1:1000) and (1:500) for primary antibody and (1:2000) for secondary antibody.
Has anybody else tried using these antibodies from Cell signaling. If yes, what amount of protein to load and what antibody dilutions gives good results?

Thanks.

[kottila]

I've used Cell signaling 1:500 for Ser 473(?haven't heard of 407) with good results on tissue, but 1:10000 on the secondary (probably not the reason for why you have no signal). What are you using it on (tissue/cells)? 40 ug is what we use as well.

Do you have a positive control?

Edited by kottila, 03 September 2011 - 05:06 AM.

[baboon]

I am using Ser 473 too(typo error in my last message). I am using this antibody on adherent  cells. Do you get a strong signal using 1:10000secondary? That's unbelievable ...Why am I not getting with 1:2000 then? I don't have any positive control..but probably will try to use that too...

[kottila]

baboon, on 06 September 2011 - 06:34 AM, said:

I am using Ser 473 too(typo error in my last message). I am using this antibody on adherent  cells. Do you get a strong signal using 1:10000secondary? That's unbelievable ...Why am I not getting with 1:2000 then? I don't have any positive control..but probably will try to use that too...

maybe everything is dephosphorylated or something? You definately should have some kind of sample you know has a high p-akt signal.

[bettan]

I've used both Ser473 (Upstate Biotechnology) and Ser474(Abcam) on tissue. Loaded 15 ug protein with a dilution 1:2000 and secondary ab 1:10000 and i got good signals. Maby its something wrong with the antibody? I have had that problem before with abs from cell signaling...

[JDiamond]

I am one of the Product Scientists for the Akt antibodies at Cell Signaling Technology.  What type of samples are you working with?  Are you including any phosphatase inhibitors in your cell lysis buffer?  

Julie Diamond
jdiamond@cellsignal.com

[baboon]

hi,

I am working with prostate cancer cell lines, loading 40ug of protein in each well. I am adding protease inhibitors to the lysis buffer.

[JDiamond]

baboon, on 30 September 2011 - 06:23 AM, said:

hi,

I am working with prostate cancer cell lines, loading 40ug of protein in each well. I am adding protease inhibitors to the lysis buffer.

Hi baboon,

40ug should be enough if there are high enough levels of P-Akt present.  In most cases cell lines will need to be treated to induce phosphorylation of Akt.  Do you have a good positive control?  Also, I strongly recommend that you include Ser/Thr-phosphatase inhibitors in your cell lysis buffer in addition to protease inhibitors.  I normally use beta-glycerophosphate and sodium pyrophosphate.  If you would like to troubleshoot further you can email- jdiamond@cellsignal.com

-Julie