Maybe your resolving gel has too high concentration of acrylamide - if so, the proteins with molecular weight higher than 80 kDa do not had the chance to go into the resolving gel - they need much more time for that.

What is the percentage of your stacking and resolving gels?

I have done thousands of SDS PAGE gels and they were always fine.The problem occurred just recently. I use the same buffer solutions that worked before. Nothing obvious has changed. The stacking gel has a concentration of 5 % and the separating gel is 10 %. My guess is that there is a problem with stacking of the proteins. During the run, I see two light refracting lines with most of the prestained marker proteins between them, but some marker bands were above the upper line. Therefore, I assume that these proteins are excluded form the stacking area.

The two layers have different functions: the stacking gel is needed to concentrate/pack all the proteins in one band, so that they will start migrating in the running gel all at the same time. The running gel allows to separate the proteins in the sample based on their molecular weight

Dear Ralf Kölling

As I understand the electrophoresis, after applying the power, all negatively charged molecules start going into the stacking gel and they just follow chloride ions (which are leading ions) present already in the gel. On the other hand, glycine (which is trailing in the stacking gel/low pH, but then leading together with chloride ions in the resolving gel/high pH), at first, only follows proteins because it is present only in the running buffer above the proteins and gels. (It is, however, constantly flowing from the buffer above the gel later, but it does not destroy the separation because it is small).

The main function of chloride and glycine in stacking is performed only in the stacking gel at the beginning of the electrophoresis. Proper pH is crucial for this: at low pH glycine is a zwitterion and moves slowly (follows proteins) but at high pH it is small anion and moves fast. I assume though that the bigger problem may be with glycine than with chloride here, and this is why:

We can say that we put following "layers": 1. chloride ions (in gel), 2. proteins (in your sample), 3. glycine (in the running buffer).

Generally, your protein should be loaded to the wells in a way that they fall into the bottom of the well (you know that for sure). But if you allow for too much mixing of the your sample with the running buffer (containing glycine) you "add" some glycine to your protein sample and maybe if there is too much of glycine within your protein/sample buffer, a problem with glycine not being a proper trailing ion appears. The problem of too extensive mixing of your sample with the running buffer may be caused by pipetting or too low glycerol concentration.

If you have higher than needed pH in the stacking gel, the problem may also occur because of glycine, analogously.

Hope it helps.

Short comment to my previous answer: If we assemble the gel and pour the running buffer for too long before the electrophoresis (I guess, long tens of minutes) then we probably also have an issue of gradual diffusion of glycine into the gel and increasing of the pH in the stacking gel. This may cause analogous situation as described above.

Dear Tomasz,

I am still wondering how the leading chloride ion front forms. The chloride ions are evenly distributed in the stacking gel, so why do they line up just in front of the proteins? Are the chloride ions coming from my sample and not from the gel?

pH could be an issue. As you said, if the pH is too high, the glycine ions may move too fast, which could interfere with stacking of the proteins. But, I use the same buffer solutions that worked before, no mistake in pipetting..

I don't know whether mixing of the samples in the wells could be a problem. In fact, I switched from using a hamilton syringe to pipette tips, which do not quite reach the bottom of the wells. Could this really be the reason?

Dear Ralf,

Chloride ions are in the gel and a little of them is almost for sure in your sample. However, I think that those from the stacking gel are in majority and are really important in the formation of the ion front (too high ionic strength of the samples destroys the protein bands). In the stacking gel we have so wide pores that they do not slow down molecules, so all ions follow each other (they build a stack of anions). Thus, immediately after chloride ions, the most mobile ions go into the stacking gel. The stacking gel is narrow, so is the chloride ion front. If we use broad stacking gel we would have broad chloride front. There is only a little of chloride originally in the resolving gel as we have high pH and we do not need to add so much of HCl to Tris base in this case. Anyway, some chloride ions are below chloride front. Thus we have chloride ions in the front and below the front. And we have glycine in the front and above the front. And the front we are actually seeing as a refracting line is probably just a boundary of "phases". We can see also the line with bromophenol blue which is small and is also forming the front.

Regarding the usage of pipette tips not reaching the bottom of the wells - yes, I think that it can be the reason, because not all of the running buffer with glycine is pushed away from below the sample and part of the sample may even gently mix with the running buffer during the falling to the bottom. You should aim the situation that glycine is only above the samples before you apply the power.

Changing the gel chemistry from glycine to acetate gels with the modification running buffer which is compatible to use at tris-acetate PAGE may be helpful at this point for large proteins.