There is not enough context in your question to give you specific answer.

If your plants went through several phases of development (young seedling -> mature seedling -> flowering),  or encountered stresses (both biotic and abiotic), OR you sampled different parts of the root, or weather fluctuations were big... See what I mean?

Also, it is good practice to transiently stain the SDS gel before Western transfer (Ponceau S; or perm stain of the parallel gel) to ensure the banding/separation is of good quality: you don't want to spend several days to find out smiles all over your blot.

ALSO, I assume you loaded the same amount of protein from each extraction?

You might wanna try 2D, too, to be able to diagnose more what's at the root of the issue.

Thanks for your reply.

Sir, the cotton plants were grown up in Hydroponics media for about 4 months and I collected whole root tissue each month....first sampling done after 1 month from the date of germination, and kept those samples @ -80C. Finally after collecting 4 samplings, I Isolated whole protein and loaded on gel, then transferred on PVDF. 2nd gel(same saples loaded here), I stained and there were we can say OK bands on it after destaining. 

I did not measured protein quantity per well as it was just qualitative expt..

Pls let me know your views.

Thanks for the explanations; this sheds some light ;)

Was the tissue from 3rd and 4th isolation healthy, or just a mash? (I never analyzed hydroponics roots after 4months).

Try minimizing storage of tissue @RT after sampling, and operate all steps of protein extraction on ice (better @4C room). Also, if cannot be loaded quantitatively comparable, try at least sample same root regions, and isolate proteins from similar amounts of tissue into same volume of buffer.

Good luck!

Yes Sir, I also observed that the roots from 3 and 4th isolation were not like in 1 n 2 isolation. They turned blackish and in meantime, there was some natural stress of white flies. Also I was not sure when n how much to feed the plants in hydroponics media.

But due to these reasons, will my protein of interest change its structure, I mean I got bands of different size........using the same antibody...?

I guess you cannot exclude some stress-related aggregation of proteins, or maybe even apoptosis due to the same reason. Or maybe even protein re-routing for utilization (either partial degradation or post-translational modifications for decomposition). Seems the samples 3 and 4 will not be reliable anymore.

Mutiple band is caused by 1) the nonspecific binding of the Ab to the sample; 2) your sample is degraded and the Ab can bind to the mutiple degraded fragments. You might need to change the blocking condition, or use another Ab. And when you extract your protein, be sure to avoid the degradation.  

Thanks for your valuable reply.

But I want to share that I isolated the protein of all the samples (1st month,2nd,3rd n 4th month old whole root ) at the one time and loaded accordingly.

I got band of expected size in 1st n 2nd month samples.

But in 3 month sample, the band size increased along with the 2nd faint band of expected size.

Then in 4 month sample, I got 1st band of increased size and 2nd band of smaller than expected size.

I mean to say that if protein is degraded, then I would get bands of smaller than expected size.

Also the antibody is picking exact sized protein band in the +ve sample (bacterial lysate) with little bit trailing, but I observed this from long time. Earlier I did this PAGE with about 3 month old plants but never seen this kind of result.

Hope you got the point that I want to convey. ...