I see 2 possibilites. First, you have 2 forms of the protein detected by your antibody...you may want to give shorter exposure when you develop the blot, and see if one goes away. If lower band is degradation product of the upper band, .You can also  add more protease inhibitors and see if it goes away.  If not, you can change the % acrylamide in gel and get better separation between top and bottom band.

But, the 2 forms maybe interesting (phosphorylated and unphosphorylated forms).. If you want to prove the 2 forms really exist, other experiments can be done. Hope this helps!

Its happened for me as well...i increased the amount of ECL for gel visualisation and also i used fresh transfer buffer....its solved...pls try

Hi Sinem, it could be related to a few things, but I would need more details of your Western blotting.

1. Are you using nitrocellulose or PVDF membrane?

2. Is it wet transfer or semidry?

3. Are you using films to acquire the ECL signal or a (cooled) digital (CCD, CMOS) camera?

My impression is that you have a problem in transfer, but on nitrocellulose it is always more grainy due to the cellulose mesh, which is larger than PVDF (for this reason PVDF is suggested for smaller proteins). Often semidry transfer produces this grainy bands due to micro air bubbles that difficult to remove, so we prefer wet transfer, also for small proteins. But you might also have acquired images digitally at low resolution or with a binning (3x3, for example, but it might be hidden in some word in the selection menu, like "sensitivity" or "high sensitivity" settings compared to "resolution" or "high resolution" settings), thus increasing pixel size.

I used PVDF membrane, wet transfer and ECL signal.

I always use fresh running amd transfer buffers. Can it be derived from activation of membrane via methanol?

Thanks for your concerns.

What imaging system are you using?  Is your camera in focus for detection?  HO well was your "sandwich" rolled etc prior to transfer?

Hi again, I think I agree with Samantha's last point, the "sandwich", since besides the grainy aspect you also have non uniform bands, more intense on one side or missing a last edge...

The activation of the membrane is easy, a few seconds in methanol are sufficient, then you can place it in transfer buffer and it's ready to be used. But when you set up the sandwich you have to make sure you press well the membrane onto the gel (or vice versa, depending in which direction you set it up) by rolling a pipette from the middle towards the edges, so to push out all (micro)air bubbles, in all four directions (center to top, center to bottom and center to sides). Push gently but firmly while rolling.

Good luck.

When my protein is in low abundance and I have to perform long exposures (with ECL and a digital imager on PVDF), the image comes out blurry. When this occurs, we use Femto grade substrate and this usually makes the image appear crisp.

Try to use an extra sponge in the "sandwich" to make sure the gel is tight against the membrane.

Actually, it does not look that bad, no worries. Does the result help you to answer your scientific problem? Why would you want to spend lots of time and resources "optimizing" the results in order to win a beauty contest?

Hi, you might try to increase the exposure time of the blot. (of course keeping in mind that you do not oversaturate the bands). also, make sure that your blocking is fine (I usually do 2 hours blocking with 5% skimmed milk powder in TBST) at room temperature. then washing the blots in between primary and secondary antibody incubation is also very critical (I wash it 10 minutes X 3 in TBST).

Hope this solves your issues.

Best

Nehanjali

Hi Sinem

I was wondering if your protein of interest is two bands. if so, I recommend 2 things:

1- increase the percentage of acrylamide.

2- increase the running time.

these will help to separate the two bands. 

please do ask me any further issues you have.

Best wishes

Shilan