I have never had problems with high salt or imidazole myself and my samples usually range from 0 to 0.5 M imidazole with 500 mM NaCl. I have even run samples with 2 M NaCl + 1.5 M NaBr on gels (but these didn't have imidazole in them) and they tended to look a little bit smeary compared to the low salt samples, but other than that they were fine (definitely no zig zags in my experience).

My guess is that your samples might not have been sufficiently denatured (incorrect loading buffer composition or not heated for long enough), which can also get worse if there was no DTT in your loading buffer. If some of your proteins oligomerise and there is no reducing agent and proper denaturation, then they'll form multimers, partial multimers and aggregates that will give you extra, unexpected bands and smears. Even if your protein of interest doesn't need reducing to run as a monomer on an SDS PAGE gel, there are other proteins that co-elute with your protein in some of the washes and these might need reducing so as not to spoil the gel. So always add fresh DTT to you samples after you add the loading dye (I don't tend to add DTT to my stock of loading buffer as I keep it at room temperature and the DTT would go off after a few days) and then heat for at least 5 minutes (some commercial buffers only need 3 minutes and the samples look worse if you heat them for too long, so read the manufacturers instructions).

BTW, I would love to see a picture of that zig zag gel, if possible. :o)

Hi,

yes, it`s caused by the high imidazole and salt concentrations. You have to remove the salts and denaturants before loading samples onto gels. There exist different methods, e.g. dialysis,....a very fast and simple method is the use of StrataClean Resin (Agilent Technologies) where you incubate a small aliquot of your sample (40 µl) with 20 µl of the resin, mix it, spin it down, wash it with ddH2O, spin it down again. Salts remain in the supernatant and so you can load the resin with bound protein (after denaturation in SDS loading buffer) onto your gel - works fine!

Good luck!

I have never had problems with high salt or imidazole myself and my samples usually range from 0 to 0.5 M imidazole with 500 mM NaCl. I have even run samples with 2 M NaCl + 1.5 M NaBr on gels (but these didn't have imidazole in them) and they tended to look a little bit smeary compared to the low salt samples, but other than that they were fine (definitely no zig zags in my experience).

My guess is that your samples might not have been sufficiently denatured (incorrect loading buffer composition or not heated for long enough), which can also get worse if there was no DTT in your loading buffer. If some of your proteins oligomerise and there is no reducing agent and proper denaturation, then they'll form multimers, partial multimers and aggregates that will give you extra, unexpected bands and smears. Even if your protein of interest doesn't need reducing to run as a monomer on an SDS PAGE gel, there are other proteins that co-elute with your protein in some of the washes and these might need reducing so as not to spoil the gel. So always add fresh DTT to you samples after you add the loading dye (I don't tend to add DTT to my stock of loading buffer as I keep it at room temperature and the DTT would go off after a few days) and then heat for at least 5 minutes (some commercial buffers only need 3 minutes and the samples look worse if you heat them for too long, so read the manufacturers instructions).

BTW, I would love to see a picture of that zig zag gel, if possible. :o)

Just to second Antonio here, I've never had problems in SDS-PAGE with high imidazole/high salt samples from IMAC columns (or IEX/HIC for that matter). It would be great if you could post a picture of the gel in question.

What a strange behaviour? I'm agree with Alejandro that it could be an help to have a picture of your gel. I nerve had problems with high imidazole (up to 500 mM) or high NaCl concentration (up to 1M). However, with KCl containing buffers, you may have precipitation of KCl/SDS that may perturbed the antry of the protein in the gel and the migration. Another explanation could be that you applied too high current to your gel that resulted in an heating of the gel, gel distortion and also bands distortion too...

What is the appearance of the front? Is it deformed too ?

What is the appearance of the Molecular weigh marker (which may obviously contain no Imidazole/no salt)? Is it deformed too ?

Thank you all for your suggestions. I will post the gel picture here. The marker too get distorted. It becomes narrow and pushed outside (loaded in 1st lane). There is another problem, The flow through samples showed bands but imidazole containing samples did not. Although they showed intense color change with Bradford reagent during elution.

I do heat denaturation for 5 mins at 95 degree celsius. I use 4x loading dye which contains beta mercaptoethanol instead of DTT, trisCl, SDS, Glycerol, EDTA and bromophenol blue. 

It may be that your protein is big in size or there may be due fluctuation in the current it follow zig zag pathway. M not sure but this may be the reason.

Maybe not the proteins but the gel is the problem? Especially if you say, that the marker also runs strange. Did you cast the gel yourself or is it bought pre-cast? Did you repeat your SDS-PAGE?

To all of you, here is the answer I sended directly to Gaurav after receiving pictures of his gel:

"I had a look on the pictures you sended. I understand why you are worried but what is the result of the SDS-PAGE i.e. what about the appearance of the bands after Coomassie blue staining ? or western-blotting and immunodetection ?

As you can see, there is some dye remaining in the wells in the top of the gel. It should correspond to protein precipitates resulting from KCl/SDS precipitates (I am now not very keen of this hypothesis) or from a too long heating before loading. Boiling samples for 5 minutes should be too much for some proteins (e.g. hydrophobic ones). You can try to heat samples only for 1' at 96°C or for 10' at only 30°C (you are working with almost pure samples so there will be probably no problems with proteases).

As the marker is also "zig-zaging", I again invite you to check your power supply and the voltage/intensity you applied to the system. Try to migrate slowly like with about 100V max for the system and 20 mA max per gels."

Now - after Gaurav provided me the pictures of his gels - I also would suggest that there (additional to high salts) exists a problem with electrophoresis itself (additional to high salts). Hand-made gels or precast? As Cedric mentioned - check the running conditions (voltage/intenisty) - gel seems to have a heat problem, too.

Hi Gaurav, 

Would you mind posting the gel image here? 

Thanks.

Well there could be multiple things . 

one is high salt . 

fluctuation in power supply. 

the voltage at which you run your gel because uneven heat transfer can also result in patterning bands. 

the running buffer needs a replacement .

Hi Gaurav, thanks for sending me the pictures, they really are more informative than a short description. As others have mentioned, it does look like a combination of problems. The fact that a band remains in each well means that the sample contains a large proportion of denatured, aggregated protein. As Cedric mentioned, certain proteins don't like being heated for too long. Also, sometimes samples start to precipitate right after Ni-NTA as some proteins aren't stable in high imidazole conditions, so you need to lower the imidazole concentration of your eluate straight away for these particular ones. Overheating the gel is another reason for strange-looking gels and is the usual culprit for the "smiley face" gels some people get. Running them at a lower voltage for longer will give you a much nicer gel. Your bands look very thick (Gaurav provided me with several pictures taken at various time points while the gel was running) and I think you might have overloaded the gel. Too much protein loaded onto the gel produces either a big smear down the entire lane or fat, blobby bands that can distort the shape of thinner bands in neighbouring lanes. Finally, and I think this is the main problem with this gel, it looks like it wasn't cast very well and there are too many ways of ruining a gel to mention here. Gels are best used on the day they are cast. As the gel ages it will shrink away from the glass panes and if you use such a gel the protein sample seeps into these pockets while running down the gel, leading to those smears you can see clearly on the surface of the gel rather than running all the way through it. This is something that happened to me a few times when I first started making my own gels, as I used to make several gels in one go to use over a few days rather than make the gels on the spot when I needed them, which is what I do now.

optimize your protein conc. and correct your power supply and run time (slow will be better)

Thank you all. The gel was freshly prepared by me (30 mins old). It's a 12%  normal gel. I ran the gel at 100 volts. I think. it's the presence of too much salt in the sample, or extra volume of sample loaded (30ul) or 5 mins heating is too excess for the proteins which have lead to band distortion. 

After staining with coomassie blue stain, I got the bands of flow through, and supernatant samples only. the markers are also visible separately but they were narrowed by the presence of imidazole containing samples on both sides. Other samples did not show any bands although they showed intense color change with bradford reagent during elution. Strange!!

 I am going to make some changes by lowering the sample volume and by lowering the time of heat treatment to one minute at 95 degree. 

@ Saranpal-  here is the gel picture image..

the first two lanes contains non treated flow through and lysed sups. marker is in fourth well while rest contains different concentration of imidazole. 

Is it a hand-made gel?

if so, can you post a picture of it?

thanks

Check your Solutions. 

Acril-bisAcril Solutions, PSA and TEMED as well. Seems to me it's a polymerization problem. PSA must be fresh and dont add more than 5% of TEMED. And check you lab temperatura also. Polymerization  doesnt like cold rooms. ;)

Hope it's help!

Marcelle Pegurier

From the photograph it looks as though the gel isn't resolving at all. Did the markers run ok or were they bunched up like the sample?

The obvious thing is to check the polymerisation and the quantity of AMPS and TEMED as others have suggested.

Do you have a stacking gel on top of your resolving gel? A 4 or 5% resolving gel at pH6.8 would help this.

Also check the pH of your resolving gel. Should be around pH8.8. 

please check pH of your buffer, specific stacking buffer, if they dont have problem, you can exchange your sample solvent,  I had same problem and this relate to pH of my stacking buffer.

Thanks for posting the image Gaurav!!

If the level of separating gel is not proper after pouring in the gel cast, bands formed may be malformed.

Thanx alot for your suggestions. It was the high salt concentration that caused the zig zag motion of the protein. The charge was not uniformly distributed during migration.