Hi

Optimum Voltage and time are gel dependet, means the % of the gel.

For this range of 25kDa - 65kDa I use 12.5% gel with 15min 75V and than increasing to 200V for 30-40min.

Good luck

Hi Waqas,

When you perform a migration of protein sample on a SDS-PAGE gel, what is of paramount importance to achieve a good resolution is the concentration in acrylamide of your gel; it depends on your protein of interest. You can easily find charts giving you the gel's concentration/resolution-range on the internet.

You have to run your gel until the migration front is at the very bottom of the gel or slightly out of the gel. It is only after a full migration that you'll achieve a full resolution of your sample.

The higher the voltage, the less time you need to run your gel. As simple. However if you run your gel at high voltage, the gel will heat up. If the gel is too warm the migration won't be nice e.g, smiling of the gel, with the lanes on the sides migrating slower than the one in the middle. Therefore, in order to have a nice even migration, it is better to run at lower voltage for a longer time. For example, you can run overnight at very low voltage.

When it comes to choose the voltage, it depends on the thickness of your gel, its size and your migration chamber.

But remember, the good migration is until the migration front has reached the bottom of the gel (the migration front can be visualized by the bromophenol blue contained in your loading buffer). Whether it takes long or not is up to you.

I hope it helps

Hello,

taking into account the PM of your proteins I would make a 10% stacking gel.

In those conditions I limit the amperage to 20mA per gel and limit the volts to 150v.

As the proteins are resolved, it will gain voltage until it reaches 150v and the amperage goes down automatically.

regards

Voltage will depends on the electrophoresis camera form and configuration, you should limit the amperage to 1.5-30 mA per well, voltage will be different, depending of the wells numbers, size of the gel and % of the acrylamide gel.

Between 12-15 % separation gel, it should works for 25 to 65 kDa proteins

Thank you Buchs Natasha, Cyrille Girard , Dolores Moreno León and Nicolás Villegas-Sepúlveda for your nice suggestion.

I utilize your valuable suggestions and successfully perform the gel electrophoresis and westron blotting, and i found that for 25KDa and 65KDa proteins on 10% gel, the optimum time and voltage is 50V for 30mints and than increasing to 150V for 50 minutes.