Sample Application Isoelectric Focusing

We got the following question:

I reversed the position of the gel … Everything went right in this experiment. The bands were extremely clear and the bands from the marker were straight … I would like to ask you if the result obtained is the effect of protein migration from higher to lower ampholyte concentrations.

The SERVA TechServ answered:

In IEF, it doesn’t matter where you apply your sample. The protein always runs to the isoelectric point. The main factors are mobility and solubility of the protein. These are influenced by many factors, e.g. buffer, pH value, hydration shell and much more. Perhaps your protein migrates better because the pH value / the ampholytes are more suitable at the new application point and the protein therefore migrates better into the gel and/or obtains better mobility …