I would assume first it's related to the protein expression in the cell lysate used. However, Just a after a quick look, Looking at the ladder which looks pretty disoriented and wavy itself on the higher bands but gets straight and sharp downwards, I would suggest, it's a transfer procedure that went wrong but that could be resolved if its the reason or not by staining with commassie blue before transfer, However, if even with commassie blue the wavy bands still exists, check your gel composition ration and mix it well before polymerization. Moreover, check acrylamide solution, it's the usual culprit with gel issues. Hope I helped!

I agree with Abdalla Abdrabou, check your acrylamide solution (it must be fresh) and wait long time for polymerization( 45-60 min is enough)

The narrowing of the lane is caused by too much salt in your sample.

Thanks for the answers so far, I am using pre-cast gels that are well within their expiration date.

I already got this problem of narrowed lanes on SDS-PAGE with my cell lysate samples and it was mostly because of the DNA remaining in the samples. Using a DNase could solve your problem.

I still think it is simply too much salt in your sample.

The many salt ions which are not impeded by the gel destroy the electric field, at least for a while until they moved out of the gel. This leads to fuzzy bands and to this funnel effect.

It is not the transfer or the gel, as the samples on the right look OK. 2 issues. 1- the lanes on the left look overloaded. Too much total protein in a lane can overload the gel and cause smearing. Depending on the thickness of the gel, it can only handle so much protein per lane. This could also be DNA, which is high MW and can cause smearing. 2- There is a difference in the salt concentrations between samples on the left and right, or something in the samples is not compatible with the running buffer and this equilibrates over time as the gel runs leaving the top looking better than the bottom. Also, Ponceau S is not sensitive at all, suggesting to me that the right is overloaded for this gel that may be too thin for the amount.

As others have mentioned, it looks like a problem with too much salt in your sample. Have you tried to clean up the sample with gel-filtration (zeba-spins, e.g.)

To reduce the salt in your samples, without having to perform gel filtration, etc. try the following - does not always work, but saves a lot of work and time when it works:

Run the gel initially at a low voltage, ~20% to max.30% of your normal setting. The voltage will be too low to move your proteins, but the salt ions will start moving. The protein, as indicated by the blue dye, may concentrate into a thin line, but if you see the dye entering the separation gel, reduce the voltage further. Run the gel with this reduced voltage for at least 30min, then set the voltage to the normal value.

If you are lucky, sufficient salt will have been removed from the samples and your gel will run and look normal afterwards.

The method does not work, when the samples are having a huge amount of salt; e.g., high molar ammonium sulfate precipitation. But your gel picture does not look too bad. It could work.

This has to do with too much salt in your samples

Is your sample look homogeneous? If not, I suggest filter it through low molecular weight (for ex. 5K, assuming you are not interested in them) cut off microfuge filters to remove in debri, insoluble material, salt, and unwanted low molecular weight proteins in sample.

I have experienced this problem and noticed that when the samples are concentrated and total protein is a lot, the sample can not move on the gel properly. You should dilute samples and run it again

Dear Melville,

It could be due to three reasons; 1.improper voltage disrubution 2. too much protein loading in the wells 3. improper polymerization.

Hi Mel,

It looks like there is another factor influencing each lane, the most likely candidate is contaminating DNA that is likely to affect the viscosity i.e. why you have varying widths. Others have suggesting this as well and using something to remove genomic DNA, the likely culprit, will help. I do not think that it is too much protein as this leads to smearing and lane widening from top to bottom. Also variable salt concentration would influence migration and this could also be involved as it affects the widening of lanes. I would recommend removing genomic DNA first, but removing salts would also be a good ideas. So that all samples are under the same conditions. Hope this helps.

Landon

Hello

Yes , i agree with Mahdiye Zaboli ; your samples have a lot of protein

Good Luck