Pedram

how can we guess, if we do not know what protein are your talking and what antibodies are you using? Observe the actin signal and imagine how much can be expressed your protein.

cheers

My protein is an oncogene that is over expressed in MDA-MB-231 cell line and I don't know how to detect that.I did many SDS-PAGEs and Western blot but all of them were unsuccessful for my protein of interest.

My primary and secondary antibodies are from santa cruz co.

This can be related to many things: cell lysis, SDS Page or blot conditions, antibody detection site, Protein size, quality of antibodies....

my lysis buffer contains:5%SDS,triton-x100,Tris-Hcl(100mM),Nacl(150mM),EDTA(5mM),sodium deoxycholate(5%),glycerol(10%).

My protein size is about 68 kDa and I transfer with NC and PVDF.My antibodies as I noticed before are from santacruz company,USA.

Sometimes Santa Cruz will provide a control protein.  Spike your cells with the control protein before extraction and run a lane of "naked" control protein.  If your experimental lane is negative and both positive controls are positive, your cell line is not what you think it is.  Please show images.

Hi Pedram,

What did you use for the blocking step? In which host was the antibody produced?

Asking because if your oncogene is a phospho-protein you should avoid blocking with milk/casein derivatives.

tnx michael.I checked with control antibody but not with a control protein.

Hi Sebastian

Tnx for your consideration.

I use 3-5% skim milk for blocking.and also I use rabbit antibody for the primary and goat anti rabbit for the secondary.

How should I know that it is phospho-protein?

As I know it has ubiquitin ligase activity

Is thre any info in the literature about this oncogene being phosphorylated. If so it is a phospho-protein. The most active blocking component in the milk is casein, which is itself a phospho-protein and it may trick the ability of your antibody to recognize its target.

As general procedure when I get a new antibody I test it with different blocking agents and in many cases I did found clear differences among them. Try also blocking with BSA 3% or other albumin based buffers and incubating the antibody overnight 4C just in TBS-Tween 0,1%.

Tnx dear sebastian

Try sampling your lysate within 1 hour of treatment with 10 minutes interval (if you're running a time-course experiment).

what do u mean Nor Azfa?

I do think we need to check on several steps here.

Did you measured protein conc extracted before?  How was it?

After transfer to membrane, did you checked either proteins were transferred to membrane via ponceaus maybe?

If until transfer process, no problem found, then you might need to play around with protein incubation period, mostly primary.  Also consider try different blot solution with diff conc.

Sometimes, when I got problem such as this, I try different protein from other brand, it might possible to get trial one.

Wish you best

Hi Pedram,

Maybe you could add proteinase Inhibitor in your Lysis buffer(PMSF and  PIC). For the Transfer condition, I guess 300mM,90min is enough to detect your Protein.And use 5% BSA for blocking .And also decrease the ratio of dilution for primary antibody.

And if it is still unsuccessful. Perhaps you could find some papers related to this Protein localization and functions, check how the other detect it (Lysis condition,antibody...) as well.

Tnx Mohammad.Its concentration is about 0.3-0.7 mg/ml with bradford assay.

I always see the ladder that is transferred,so we can assure that transfer condition is OK.

Hi Yanxin.tnx

I use PIC from sigma company and my lysis buffer is suitable to extract any protein from cells,without any regard to its localization.

Hi Pedram, I was reading the comments you've got so far, and I was wondering how do you prepare the samples you load in your gel. I had a similar problem with a transcription factor and I realized that the protein degraded when I boiled the sample. So, if you haven't tried it yet, maybe you should give it a shot. Hope it helps. Good luck.

Last time when I tried to detect a phosphorylated protein in a time course experiment (even when most parameters has been optimized), I failed to get a signal when the sampling was performed after more than 1 hour of treatment with drug. Someone reminded me of how fast the phosphorylation could occur and suggest to perform the sampling within short duration of time after drug exposure. So, I did the sampling after 10 minutes of exposure followed by 20 minutes etc. until 2 hours, then only I managed to get the signal. It turn out that for that specific phosphor-protein I targeted, the activation was very fast, possibly within seconds after activation. But, this may be the last option after you have optimize or try other possibilities suggested by others. 

Hi Pedram, if your protein of interest is a phospho-protein, you would probably require a phosphatase inhibitor in addition to your PIC. Also, like what Nor Azfa Johari had mentioned, treatment time could be essential. Perhaps you could verify with a loading control as well.