Never rely on a single phospho-specific antibody before ensuring its detecting what you think it is. The Cell Signaling Technology antibodies are usually among the best but much depends on aspects unrelated to the antibody including the lysate preparation and secondary antibody, not to mention the expression level of the target and whether it is being phosphorylated. In the case of Akt/PKB, as others have mentioned, you should perform a total Akt/PKB immunoblot - this is an essential control in any case. You can also try the phospho-Threonine 308 antibody as both sites are typically induced upon activation of PI3' kinase. Run controls from cells where you know the pathway has been activated. 293T cells are easy as they have defective PTEN and so tend to have high Akt/PKB phosphorylation even without agonists. Serum starvation followed by 10% serum is also a generic way to stimulate the pathway (but its better to use more defined agents such as IGF1).

Note, most phospho-specific antibodies are not as good as those available for Akt/PKB.

Which blocking buffer are you using? We collect proteins in our cell cultures with RIPA-BUFFER (Contain protease inhibitor coctail)

We don't face any problems with collecting and imaging system

Can you share your protocols?

If ı use this blocking buffer Does it give multiple band?

This is one of the good antibody produced by cell signaling and it will work at any worst condition. If you directly add on the membrane after transfer in TBS still it will work. I think you might have problem in transfer or phosphate se inhibitor cocktail or isolation.

Do you have a positive control, e.g., stimulated cells?

Maybe too simple: did you use the correct secondary antibody?

The antibody should definitely work, i.e., if you have a p-Akt....

There are a lot of possible explanations for your problems. You have to give more detailed descriptions of what you're exactly doing.

1) what cells are you using? are you sure that there's P-Akt in these cells?

2) Nobody's using just P-Akt abs. So what do you get when you stain for total Akt? Are there any Akt in these cells?

3) It could be a problem with your lysate. We routinely lyse cells in 1xSDS-PAGE sample buffer, it's the best way to make sure that all your phospho-proteins are still phosphorylated, and you dont have to add any inhibitors. But it is suitable only for Western blotting analysis, not for cases when you need native protein lysate

4) Does your Western blotting protocol work with any other antibodies?

I have found out that these tips from Abcam are very, very good and true, especially the second one, which most of the people forget if they have milk in the protocool for blocking solutions or special blocking buffers, e.g., from Roche (it is mostly casein):

1. Keep the proteins in their phosphorylated state! Add adequate phosphatase

inhibitors and keep samples on ice at all times.

2. Block the membrane in 5% w/v BSA (fractionV) NOT MILK (milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk).

3. Remember the phosphorylation may need to be induced. Low signal or no signal may mean that the induction is not sufficient. Run the recommended positive control with your samples.

Many of the suggestions above would probably shed some light on your situation. We've recently published a paper including a protocol using this antibody, with some detailed methodology:

http://www.molecular-cancer.com/content/10/1/76

I've heard numerous times from many people that using milk with phospho-specific antibodies can lead to a high background from non-specific staining (or really, specific staining to milk phosphoproteins, but non-specific to your proteins of interest!). I have no argument with that sentiment. That being said, for the pErk and pAkt anti-mouse antibodies that I've used, I've never once had that problem. It gets back to antibody affinity- there's alot of casein in milk, but if the antibody has low binding affinity for casein (and this is a monoclonal!), then milk can and does work just fine. And specifically for this antibody, I've done it literally hundreds of times optimizing the blotting and incubation conditions, and it works just fine. Bottom line: specifically with this antibody, the presence of milk while blotting may not be your problem. Milk during incubation? Maybe...I never use milk in the incubation buffers.

Troubleshooting: do you see any proteins, or are you otherwise sure that the transfer was complete? Can you detect appropriate levels of any other phospho-proteins, such as pGSK-3beta, which would indicate Akt activity (i.e. a functional marker of Akt)? Do you see total Akt and just not pS-Akt? Did you trying rocking at 4C overnight incubation for the primary, vs 1-1.5 hrs at room temp? Are you developing film, and if so, are you exposing long enough? In other words, does everything seem to be working except for this specific antibody, or is this just the "tip of the iceberg" of WB issues?

Lastly, there are 3 isoforms (or more?) of Akt: Akt1, 2 and 3. I'm not sure if this antibody is specific to one or several, but an outside possibility is that your cells are expressing an isoform which is poorly bound by this antibody.

This is a wonderful AB. We use it for western 1:2000 and block with milk as always (...never had problems with phosphatases in milk). However it is essential to add phosphatase inhibitors to your cell extraction buffer(e.g. 2mM sodium fluoride + 0.5mM sodium vanadate final concentration). Starve some cells for serum (4h) and then add back serum for 15min to get a positive control.

Hope that helps.

Robert

Has the Ab been stored and handled properly (Mfg suggestions, no freeze-thaw, etc.)?

I thing it is also important to see if you have enough total protein of your loaded samples(over 20 microg/well). If you have very low protein concentration you could not detect band. I also use Phos (Roche)Stop inhibitor for phospho proteins.

I have used this same antibody and it works just fine. I block with 5% milk in TBTt and incubate the pAkt antibody at a concentration 1:1000 in TBSt+3%BSA ON. Secondary is in 5%milk again at a concentration 1:5000, using Amersham secondary. It gives a nice single band with low background. I used mice, rat and human endothelial cells, and macrofages. But also liver lysates and it works well with all of them. Akt has 3 different Isoforms, and if I am not mistaken this antibody has higher affinity for Akt1 and 2, it could be that your cells express a different isoform, however I would expect a weak affinity at least, Try including some positive controls like FBS stimulated cells, or cancer cell lysates known to have Akt pathway active.

Activation of kinase phosphorylation by heat-shift and mild

heat-shock...Cell Biol. Int. Rep. 2010...we have published this paper few years ago...you will see many cell lines ...pAKT antibodies from cell signaling are very good ...it is pAKT signal that can be very variable! as well total AKT

try also to increase the amount of sample

Well, I will use positive control cell lysate to start the troubleshooting first..... make sure the internal loading control (GAPDH, actin, etc) show you the results. Strongly suggest you using 5% BSA (BSA used for immunoblotting/IHC) to block non-specific binding and prepare primary and secondary antibody. Good luck

Never rely on a single phospho-specific antibody before ensuring its detecting what you think it is. The Cell Signaling Technology antibodies are usually among the best but much depends on aspects unrelated to the antibody including the lysate preparation and secondary antibody, not to mention the expression level of the target and whether it is being phosphorylated. In the case of Akt/PKB, as others have mentioned, you should perform a total Akt/PKB immunoblot - this is an essential control in any case. You can also try the phospho-Threonine 308 antibody as both sites are typically induced upon activation of PI3' kinase. Run controls from cells where you know the pathway has been activated. 293T cells are easy as they have defective PTEN and so tend to have high Akt/PKB phosphorylation even without agonists. Serum starvation followed by 10% serum is also a generic way to stimulate the pathway (but its better to use more defined agents such as IGF1).

Note, most phospho-specific antibodies are not as good as those available for Akt/PKB.

In our lab, we noticed that when using Rabbit mAb from Cell signaling, all secondary antibodies anti-rabbit didn't work. When using swine anti-rabbit HRP (P0217 from Dako) we get no signal at all but with the Goat anti-rabbit IgG-HRP (#7074, Cell signaling) it works very well.

I don't mean that it is the only secondary antibody that will work (cf : Juan Rodríguez Vita) but perhaps it could solve your problem.

Good luck

We get a lot better signals with almost all phospho-specific antibodies when we incubate for a longer time period - up to 3 or 4 days in the coldroom. Also, we have no problems using milk with this antibody.

Agree with James Woodgett. You haven't stated what samples you are trying to measure P-Akt. If cells are stimulated with Insulin/IGF1/serum following serum withdrawal, then you shouldn't have a problem. Basal levels maybe very low, too low to detect.

Even I din get bands with p-Akt S473 #9271 (cell signaling) in mice liver tissues.

you can use MDA MB-231 lysates (without any treatment) as a positive control for P ser 478 Akt. these cells have a lot of it. I use the same antibody and allways get very good signal in these cell lysates.

Hi there... I know this is an old post but I'm having the same problem with p-Akt. I get a strong signal for the positive control but nothing else. I'm using rat heart tissue and I homogenized in RIPA buffer with several phosphatase inhibitors. Is it possible for something in my RIPA to inhibit or interfere with p-Akt? Its a cell signaling antibody and I used a 1:1000 dilution and blocked in milk. I think my buffers, gels and technique are fine because I have obtained great bands for other proteins. Please can you give any suggestions?

 Hi there i also am using phospho akt antibody from cell signalling. does anyone have any idea if there is expression in SiHa cell line..?

Hi! For how long should one stimulate cells with FBS following serum starvation to stimulate the pathway?

I used to grow them in complete media for 24hrs prior to experimentation so that the achieve morphology. and then starve them for 4-5 hrs before stimulating or inhibiting the pathway.