I would advise you not to use photoshop as Paul has stated.  It is not good to alter images using this program and is not scientifically ethical. After looking at this blot, you have some issues that need to be fixed before you can adequately asses the protein of interest.  In my opinion it is not possible to obtain any information from this blot because of the smear.

1. what form of your samples are you running?  Does this represent whole cell lysis or is it some purified sample.  To me, it looks like you need to further purify your sample to avoid the smear appearance.  Are you performing any centrifugation steps? 

2. How much protein are you loading on your gel?  It looks like you could load less protein based upon the smear.

3. What is your antibody concentration?

4. Are you blocking the blots in milk, bsa, odyssey blocking buffer?

you cannot use photoshop to improve these results Salman. It is un-scientific and not honest. You need to get rid of your excessive background by using blocking agents , less antibody or a better antibody but others are better qualified than me to advise specifically on this problem

you can use BioID or ImageJ software, by which you can specific area.

I would advise you not to use photoshop as Paul has stated.  It is not good to alter images using this program and is not scientifically ethical. After looking at this blot, you have some issues that need to be fixed before you can adequately asses the protein of interest.  In my opinion it is not possible to obtain any information from this blot because of the smear.

1. what form of your samples are you running?  Does this represent whole cell lysis or is it some purified sample.  To me, it looks like you need to further purify your sample to avoid the smear appearance.  Are you performing any centrifugation steps? 

2. How much protein are you loading on your gel?  It looks like you could load less protein based upon the smear.

3. What is your antibody concentration?

4. Are you blocking the blots in milk, bsa, odyssey blocking buffer?

I would recommend you to examine in detail the editorial "Manipulation and Misconduct in the Handling of Image Data", enclosed here as a PDF file.

Since your question concerns particularly a problem of existing strong background, I have highlighted on the first page one relevant paragraph:

"Simple adjustments, applied uniformly, to the entire image are generally acceptable. Changes to brightness, contrast, and color balance fall into this category because they affect the image in a linear fashion. However, it is not acceptable to adjust brightness or contrast levels to such an extent that image data are truncated or lost".

Hope that this information is useful, and wish you success with the further experimental work.

Kind regards,

Atanas

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Dr. Atanas G. Atanasov, PhD

Principal Investigator

Molecular Targets Group

Department of Pharmacognosy

Faculty of Life Sciences

University of Vienna

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I agree with the clear sentiments of the other responders here that you should not alter or "massage" the image via Photoshop or other software. I also agree with others that the background is high enough that it compromises the interpretation. Thus, I strongly encourage you to spend some time optimizing the use of this antibody in your Western blotting protocol. Try different blocking buffers; including Blotto, BSA, different detergents, etc. There are many different blocking buffers that can lower background but be aware that they can also affect sensitivity to your protein. You might also compare transfer of your proteins to nitrocellulose vs PVDF membranes. And once those issues are resolved optimally, I would suggest that you compare different dilutions of your primary antibody to again optimize signal to noise. These are all issues known to affect signal:noise ratios in immunoblotting results and should be done for each antibody, though rarely are. But they keep you on the right side of any ethical concerns and is best practice for immunoblotting.

Dear Salman,

As you can see from the comments here - using Photoshop is in general viewed negatively from the scientific community: the reason is that it is a very powerful software package, and indeed one can use it in many beneficial and legitimate ways (e.g., for image resize or for file format conversion), but it could also be easy abused, resulting in scientific misconduct (e.g., for selective processing of a defined area of an image). This is the reason why I recommend you to really inform yourself very carefully for the good scientific practices in this context, if you intend to ever use Photoshop for any science-related applications. For this purpose, the editorial of Dr. Martin and Dr. Blatt:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3809520/

...might be the best scientific resource on the topic (to the best of my knowledge).

Concerning the particular image that you have posted here - I assume that you have (or plan to make) also pictures from other independent experiments (since one needs at least n = 3 for a publication)? In line with the opinion of the other colleagues who commented on your post - it is appropriate that for the future experiments you try to optimize your protocol in order to reduce the background. Aside from the suggestions that were already made here,  some further possibilities for background reduction are also listed on these sites:

http://www.abcam.com/protocols/western-blot-troubleshooting-tips#high%20background

https://www.abdserotec.com/western-blot-high-background-signal-on-the-blot.html

http://www.rndsystems.com/tsg_detail_objectname_western_blot.aspx#4

Kind regards,

Atanas

--

http://homepage.univie.ac.at/atanas.atanasov/

Looking at your blots definitely you need to repeat the assay. The dark spots diffused in several point are usually indicatives of inadeguate blocking step of the membrane .Are you blocking the blots in milk, bsa, or other buffer? For how long?  A less concentrations of proteins could help in a less smearing and a more clear bands.

A little less exposures time can help also to look better at the bands.

But it could also be possible that some degradation is in place ( high intensity of lower bands ). So it is important to know exactly other details of your experiments.

Definitely not modify the figures of real results.

Regards

Maria Vitale