I have seen the artifact in lane 9 occur when a specific protein within that sample has reached it's pI and precipitates within the well. The cross contamination of your marker lane makes me wonder how old this gel is and if the stacker has detached from the plates on that edge. I too agree with dilution of those first 5 samples about 4 fold with 1x reducing buffer.

it is possible that your gel concentration in terms of SDS or Agarose is not high enough leading to the pores being too large and not separating the molecules accordingly.

Also hope that a stacking gel is used? if not look up a protocol for the stacking gel, this will allow the molecules to all start sepArating at exactly the same time.

Yes, of course I'm using stacking gel also. 

It is the result of AKTA purification on Talon resin. I looked at the profile of purification and maybe I have found the answer. The fraction's absorbance in 9th well is 10 times higher than in the other wells, so maybe it's just overloaded.

I have ready-to-use samples to run SDS-PAGE , but I'm wondering now if i can dilute it with water in 1:10 ratio to get  an appropriate resolution? 

First, your sample concentrations are too high.  Dilute down.  Second, if making your own gels, I agree with Hussein Fawaz.  If purchasing, make sure its not past the expiration date.  Other ideas, running buffer to gel compatibility and running the gel at the right voltage to avoid overheating.

I agree with David Arginteanu you are loading an excess of protein to each well so dilute your samples,  also it could be that the running buffer is warming up while running.

agree with David too

Thank you very much for help! I will try out all your recomendations tomorrow :)

Can I dilute it with water, or with some other agent?

I have a doubt that , Have you loaded the same sample in the 9 to 12 lanes? If yes their might be a smile/ air bubble in your gel while making. Try to avoid air bubbles while making your separating gel. Because the only lane got the problem and also so many bands appear in the lower molecular weight. I hope this will help you. Check your protein concentration before the SDS- PAGE analysis and load the sample accordingly.

Besides the reasons described above, your sample might contain too much salt(s).This will interfere with the separation process.

If this is indeed the case you can perform acetone / ethanol precipitation to clean up the samples (those methods can have rather high recovery rates, >90%). The pellet can be dissolved in laemmli solution followed by SDS-PAGE.

In my experience, this kind of smear sometimes comes from DNA being present in the sample. If this is the case and your sample is very viscous, try adding a little bit of DNase and incubating 5-10 at room temperature or, alternatively,  to breake the DNA into smaller fragments by freezing and thawing the sample several times before loading, vortexing in each cycle.

As Patricio mentioned, residual DNA in the sample can also smear the lanes. In addition to DNase one can sonicate the sample. Even very brief 3x10 sec bursts with 10 sec pauses in between would be enough to achieve DNA disruption and can give well resolved band patterns.

Just centrifuge these samples for longer time 12000 rpm for 20min and load the upper layer am sure your problem will be solved.

Some of the lanes (4,5,6) seem to be overloaded. Dilute and centrifuge before loading.

I think this is not due to overloading, overloading is evident in lanes # 5 and 6. This can be due to the presence of any insoluble particle in the gel or a trapped air bubble, which you can see as a circular pattern. This can be because of DNA in the sample as mentioned by worthy scientists, but the point is why only in one sample. You should repeat same sample in new gel and see. If the problem persists, you should think about DNase treatment.

Good Luck.

Lanes 4,5, 6 and 9 are massively overloaded. If it were because of just one protein (as happens when running serum which is half albumin) it can be removed perhaps by affinity chromatography (Affi-Gel blue is great for albumin), so that the other proteins can be visualized more clearly. The smear in lane 9, especially if this is a higher concentration than lane 8, may mean you have some type of polymer perhaps with carbohydrate moieties that isn't fully dissociating; if you overdose the loading buffer with too much sample, this might be an effect. But lane 9 is such an anomaly, perhaps there was a gel artifact in that lane: look at the mushroom-like deformation at the top, and it looks like the sample had to detour along the sides around something blocking the center of the lane.

Samples might be dialysed to remove salt that smears all patterns, but other bands look good-- doubtful salt is an issue here.  You have so many proteins in such close bands, it would be good to run gradient gels to space them apart for clearer resolution.

The pale staining overall indicates incomplete destaining, which needs to be done with at least three fresh changes of destain. DNA is not an issue.

is overloaded you should  diluted and warm longer and to avoid cross-contamination between the lines  the height of the pocket must be larger

To be honest most of the gel looks fine to me! As others have pointed out, some of the lanes are overloaded and it's a simple matter of dilution (into the buffer that the protein is already in) to obtain better resolution. The artifact in lane 9 is weird. It can't be an obstruction or airbubble in the lane because that would affect all bands and most of them are fine. Unless the obstruction/bubble appeared later in electrophoresis, in which case that's just bad luck and unlikely to happen again in the same spot. The simplest solution is to run the gel again with diluted samples.

Try running it slower, to optimise resolution. Hope it helps.

I have seen the artifact in lane 9 occur when a specific protein within that sample has reached it's pI and precipitates within the well. The cross contamination of your marker lane makes me wonder how old this gel is and if the stacker has detached from the plates on that edge. I too agree with dilution of those first 5 samples about 4 fold with 1x reducing buffer.

Dear Malwina,

      I agree with Cynthi M. Rodenburg. pH of the buffers matters, at the same time before loading the samples centrifuge at high speed for two minutes and load the top layer. After observing your gel i can say that your running buffer is old it is not fresh so run the gel in fresh buffer so that you can avoid the aggregations of your protein and other contaminants.

  All the best. 

Malwina, regarding your question on how to dilute, in many cases simple distilled water will cause problems-- you want it as best dissolved as it can be before it hits the stacking gel, and the lack of all ions in the solvent can cause precipitation (you would see scum at the interface of the well and gel). Here's a basic recipe of a concentrated sample buffer that you then dilute with your sample--- and you will be adding a lot less sample. http://openwetware.org/wiki/SDS-PAGE_sample_buffer_%28Morris_formulation%29 

But just try loading less into the well-- if you're loading 10 microliters, make it 5. Your other lanes, although heavy, run fine, and so it isn't wise to fool with your preps or formulations.

I think in lane 4, 5 and 6 you may loaded a high concentration of ptotein. Tray to use less amount of protein with high salt and in the presence of reducing agent.

Hi Malwina-  You do not want to dilute with water, as it will throw off both the ionic strength of the sample and reduce the detergent concentration, and therefore the SDS/protein binding equilibrium.  Both need to be consistent.  If you dilute, use 1x sample buffer to maintain approximately constant ionic strength/detergent.  I suggest measuring the protein concentration rather than guessing.  The BCA assay is relatively insensitive to SDS at low concentrations, and could give you accurate measure.  In lane 9, it appears to me as though there was a gel anomaly, such as a bubble, a bit of poorly mixed gel, or something of that nature.  If casting your own gels, just re-cast a gel and re-run the samples if you still have them.  If these are commercial gels I would contact the manufacturer and provide the photo.  As the SDS gives everything a net negative charge I don't think a protein hitting its iep is too likely the problem.  In any event, there would be some (highly basic) proteins in every complex sample with the potential to do that, and the artifact would then appear in most gels, which it typically doesn't.  Good luck with your project.

Dear Malwina,

Check salt in samples, dialyze them if required.

Reduce protein concentration loaded in wells (lines 4,5,6).

Reduce running voltage, take care of ampers. An electrophoresis chamber with cooling systems will help to avoid gel distortion by heating.

Yours,

Hector Nolasco

Ideally 15 ug/10 ul concentration of total protein sample will give u clean run without smear..

The voltage used is too high and try to decrease the voltage by 25-50%.

The concentration of the protein is too high; you can reduce the amount of protein loaded on the gel.for this one you can read the concentration of your protein via Bradford then pick enough amount of sample and load on SDS-Page 

The salt concentration is too high, use dialyze the sample, precipitate the protein with trichloroacetic acid (TCA) or use a desalting column.

Your sample has high salt. Dialyze it, add low protein. Use 50 voltage for 30 min first, then rise it to 150 voltage

I would suggest to keep empty line between samples, this will help you to read gel better and to see whether your loading is correct and samples not overflow

Hello..

I suggest you note the size of stacking gel, it is important that he has a good size for the proteins start in the same time.

Other thing is about your running voltage, get a lower voltage.

Hello Malwina

Well, I think that three different procedures can help...1) Please boil the samples prior the applying in the gel, it will reduce the artifacts 2) Reduce running voltage, you can put the mA constant at 15-20 mA, it will make not much than 100-110 volts in the start of your running; 3) Increase the size of the stacking gel, usually 2-3 ml of gel is enough to 1 mm glass plates at BIORAD systems. Other thing that seems to be naive but it´s not - is to clean the wells with a hamilton syringe prior the application of the samples - because it can disrupt some unwished gel-grain which is formed after a slow removing of the gel comb. It can hind a good and sharp band formation sometimes...Hope these suggestions can improve your results....Bye Bye

A likely explanation is that each time there was a delay between loading the samples and actually running the gel might cause this problem. please go through the link below for troubleshooting 

http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gelgoofs/bizarre.html#3

I think that your samples have high salts . you can make dialysis before running the sample. or run samples at 40 volts for 30 min to remove the salts