Hi Roni,

The gel picture suggest that the gel was over loaded with the sample. Have you tried to load less sample, maybe 1/10 (or less) of what you loaded in the gel. In my experience the overloading would give this effect. If the smear was bellow your protein it would suggest degradation/digestion of the protein, and addition of protein inhibitors such as EDTA, PMSF, benzamidine would help reduce the that. Hope this helps

Hi Roni,

The gel picture suggest that the gel was over loaded with the sample. Have you tried to load less sample, maybe 1/10 (or less) of what you loaded in the gel. In my experience the overloading would give this effect. If the smear was bellow your protein it would suggest degradation/digestion of the protein, and addition of protein inhibitors such as EDTA, PMSF, benzamidine would help reduce the that. Hope this helps

Hi,

  many proteins can produce smears due to post-translational modification, such as phosphorylation.

It seems an artifact, may be the gel was too thin, the gel glasses hydrophobic. Was the molecular weight correct? Try again with a gel made of fresh. Good luck 

From looking at the gel, I agree with Vlatko that the gel seems overloaded.

There is a possibility that this is a result of post transnational modifications (PTMs), such as phosphorylation, yet quite unlikely, particularly because PTMs tend to produce two or more bands and less a smear.

I would recommend for a 1mm thick gel to load not more than 10-15ug/lane (depending on the width of the comb), and for your case estimate first the protein concentration (e.g.. - using Bradford, & BCA reagents, or measuring the OD at 280nm (using a Nano-drop for example), and run an amount as above .

From looking at the gel, I agree with Vlatko  and Ghil  that the gel seems overloaded. 

Try just to dilute it 5 and 10 time and reload it to see whats happen. 

I do not think that you have a problem of degradation because the smear is a higher mw respect that the mw of your main band. Degradation generally produce small mw fragments. 

Did you hadded any DNAse in your lysis buffer? Some times presence of high DNA amount in the sample can also result in similar smearing  (in this case is possibile htat you have also problem to load the sample in the gel wells because after boiling it is become a sort of glue). Did you observed something similar? In this case you can try to add some DNase at your purified sample and load it again. 

Any of the previous answers may be correct. It is possible that the protein quantity is too great. However it is also possible that the protein concentration is too high and this has caused some precipitation of the protein. This may occur if you are using a stacking buffer system such as the commonly used TrisHCl/Tris-glycine buffer. The stacking system will concentrate the protein prior to separation. When you try loading less protein it may be worth  testing a sample with a lower protein concentration. Another reason for smearing is that your sample may be too salty. This is another reason for diluting the sample.

Load less amount of protein, Remove any debris from sample using centrifuge, and  use protease inhibitor. surely you will get good results

Thanks for the answers. 

I tried to load less protein but the smear was still there, even though not much as before. The molecular weight of the protein is correct. 

 It seems there is incomplete denaturation of the protein because when we tried to add more SDS in the sample buffer the smear was much reduced. 

Any thought on that?

How much less protein did you load? Do you know an amount/lane?

can you add another picture of the current situation as a comparison?

Can you try a fresh batch of reducing agent and SDS? Might be worth trying if lowering the load of total protein did not solve your problem.

How much SDS do you use (I normally use 4x stock 2% LDS)?

I agree with most of the comments and answers given above. In addition, high concentration of the salt in your buffer may also cause smearing of your proteins. Did you purify your protein using a buffer with high salt concentration?

Sure, protein overloading might produce this effect and loading less AND using fresh buffers and denaturing reagents is the easiest thing to check.

However, PTMs, such as N-glycosylation and phosphorylation, especially if the protein is large and several PTM loci are present. MS techniques show how variable PTMs can be, routinely detecting peptides with or without modification and where more than one PTM is possible, combinations of these. For a globular protein with e,g, 3 phosphorylation sites, the differences in mass may be as much as 3! x 80 Da, or 0.48 kDa, with 23 intermediate masses possible (smear). You could try adding phosphatase, or examine the MS/MS spectra of peptides, allowing for N/T/S phosphorylation.

N-glycosylation, especially complex types can also vary in the degree of modification, leading to similar smears.