For this next step we will separate the individual proteins in our sample
lysate based upon their molecular weight using a positive electrode to attract
the negatively charged proteins.
To do this we load our previously prepared protein samples into a commercially
available polyacrylamide gel.
Gels are available in fixed percentages or gradients of acrylamide. The higher the
acrylamide percentage the smaller the pore size of gel matrix.
Therefore higher percentage gels are better for low weight proteins,
low percentage gels are better for large weight proteins and gradient gels can be used for
proteins of all sizes due to their varying range in pore size.
Prepare your gel by inserting it into the electrophoresis apparatus and
filling with running buffer that is appropriate for your gel chemistry.
Rinse the wells of the gel with running buffer and add buffer to the
chambers.
Load your samples into the wells. If you are unsure of the amount to
load, 10-30 micrograms of total protein is a suggested starting point as
well as tighter amount of sample loaded.
You will also need to reserve at least one well for prestained molecular weight
ladder.
The ladder will allow you to monitor protein separation during
electrophoresis and subsequently verify protein weight in your sample during
later analysis.
Close the electrophoresis unit and connect it to a power supply.
Most units typically run 45-60 minutes at 200 volts
or until the loading buffer reaches the bottom of the gel.
During this time the negatively charged proteins in each sample will migrate toward
the positively charged electrode making their way through the polyacrylamide
gel matrix.