Hi,

I am not sure about numbers of well and the thickness of gel. Usually, 1.5mm (tickness) gel with 10 well can be used to lead around 50ul of sample. If you think the sample volume is too much and probably sample is transferring to the next well, it will be wise to reduce the volume of sample by making the highly concentrated protein during protein extraction. Regarding the protein separation, it seems like the voltage is much higher in first gel (stacking gel). In our lab, we run SDS-polyacrylamide gel electrophoresis at 35-50 V till the last band of the marker (depends on the % of gel) is exposed. Then change the voltage to 130 V till end. Hope, this will help to help you to avoid the smear effect of band though it is time consuming. Another things are always maintain the level of buffer (fill up). Try to avoid to bubble formation during the gel preparation.

There can be leakage between wells. You can fill every other well with agarose to plug them, then use the open wells without lateral leaks.

I agree to Ellens' opinion, maybe there is a contamination due to a "leakage between wells".

Dear Nada,

this is a very common problem, that can be solved in at least 2 ways:

- if the cause is wells leakage, it could be necessary to check reagents' quality and expire date, as well as polymerization time and temperature (in order to be sure of the process, it also should be checked buffer's pH and reagents' quality)

- if the cause is a contamination, it could be necessary to handle the entire process more accurately; maybe a more specific antibody could come in handy

Good luck with your work,

Dave