This ooks like a loading issue perhaps do you quantify your protein first? Every antibody gives different results and sometimes you get non-specific bands. If i were you i would find some good papers using the specific antibodies you have used and copy that protocol exactly. Also by looking at papers you can see what non-specific bands are to be expected and what you should see. It looks as if you have plenty of protein so extraction is not an issue.

 Thank you for the reply.And i do quantify the proteins.i will follow what u have said.  But i follow the same protocol of my senior . She gets the results every time. And i get the clear results only sometime. I dont know where i  did  wrong.

Looks also that you have overloaded your slot,

how much protein do you load?

I think you already know that but be careful at the step of transfer. Don't touch the membrane of nitrocellulose (or PVDF) with your fingers (even with gloves), use a plier. Also, be careful with bubbles between the gel and the membrane. In your case, did you try to load less protein ? It seems that there is some trouble with the migration of your proteins. Good luck !

no i dont think protein concentration has to do something with that . becoz i load always the same concentration of protein. Some times the result is nice sometimes its not . i havent found the solution till now .

Are you making your own gel or are you using one already prepared by a manufacturer ? If your making your own gel, maybe something went wrong with the preparation...How do you prepare your samples ? Don't exceed 10% glycerol, don't forget to heat the sample at 95°C for 5 min and use buffer at 4°C. Maybe someone else have better ideas :D

Sumeet, looking at your control bands it seems that the protein concentration or loading is not a problem (i'm assuming here that these are loading controls that you are cutting out from the same gel as your test bands).

It could be differential expression of the protein of interest in different samples i.e. somehow your protein is being expressed differently in your different samples/different lanes. Though you say you have not treated your samples differently, is it possible that your samples are being unintentionally exposed to different conditions (temperature/medium/stress)? 

If you always use the same amount of protein  you could still have the combination of crappy gel and overloading, whats the amount of protein that you load?

and the percentage of gel? and how long do you incubate with primary antibody and secondary antibody.

In addition to the other ideas, I would ask about the concentration of your primary and secondary antibodies, time and temperature of your incubations, your blocking conditions and your washing buffer and conditions. It would be best to post your protocol to narrow down the questions.

First i prepare the mixer of ripa buffer and PMSF . then take the sample and scribble it. transfer it to the epitube and incubate on ice for 3 mins repeats for 3 times and invert each for 4- 5 times . then centrifuge for 10 mins in 14000rpm after that the debris is removed then assay is done after that 4 *sds is kept with b mercaptoethanol and for higer moleccular weight beta mercapto ethanol is not kept . then only the low molecular weight with betamercatoenthnol is heated for 2 min each for 3 times in the middle vortexing for 3 times and keep on is for 3 mins .and 3 mins centrifuging is done and then loading . and is use 0.1 ug/ml primary antibody and sencondary antibody 1:5000 ratio. i put primary antibody for overnight , secondary for 1 .5 hr and blocking for 1 hrs and i use 10 % SKIM MILK for washing and blocking . i wash 5times for 5 mins after primary antibody with skim milk and 3 times with skim milk and two times with tbst at last after secondary antibody.

Possibly measurement of protein concentration is not consitent over these time period.

WB could be very difficult to troubleshoot and when you get to the level whereby you are able to figure out the reasons why you have the result you have, you are actually reaching the level of an expertise. it is normal to face issues like this while just starting. You must continue until you get good result.

Now, looking at your question, there are many reasons why you might have inconsistent result. If you are working with cells, your troubleshooting starts from the cell culturing technique. The question is were your cells in perfect condition before harvest? Are you sure you are not using infected cells. Hmn! The reason is that infected cells would also give you bands if you are good at WB but it might not be consistent, what I mean is that it might not give the expected blot trend.

If you are sure of that, then you need to troubleshoot your extraction procedures. This step is also very important as you know that cells are very fragile. Are you sure you do not damage the cells during trypsinization and harvest? Are you sure your steps of protein extraction are perfect?

Looking at your blot, it may be that the loading of the protein was not proper while running SDS page. For the control, I suggest you dilute the antibody the more. it seems the antibody is just too high. The same challenge might be applicable to your desired protein but if that is the issue, are you sure you loaded the appropriate volume of protein per well

You can use the formula n=cv; where n = the number of moles of the sampl/well, c= concentration you got from your calculation curve after you've multiplied with both  the loading buffer constant and your dillution factor and v is the volume of the samples you wanna load. Therefore v = n/c. I always load 60microgram/microlitre. So, you can divide the 60mg/microL by the concentration of the sample to know the volume you will load per well. You need to load the same volume per well. You may go for the lowest volume after your calculation because all the protein wont give you the same volume but it may be very close.