Welcome to the Novus Visual Protocol Series.
In this video we will learn how to perform all phases of a Western Blot using the
most common methods for this assay.
Before we can start preparing the blot we must first prepare our sample lysate.
In this example we will prepare a protein lysate from cultured cells.
Here we wash the cells twice with ice cold PBS and enough lysis buffer
to cover the cells.
The choice of lysis buffer depends largely upon the localization
of your protein of interest.
We scrape the cells and transfer the cell solution on a centrifuge tube
placed on ice.
In order to solubilize membrane bound proteins, we will require stronger
extraction detergents compared to isolated cytoplasmic proteins.
In this example we are using a standard RIPA buffer, which is a common buffer
for obtaining maximum protein yield. While extracting proteins from all
cellular localizations,
it is very important to include protease inhibitors in your lysis buffer which will
prevent degradation of your sample. Always use freshly prepared protease
inhibitors, keep samples on ice and work quickly.
We lice the cells by pipetting up and down
followed by incubation on ice for thirty minutes.
Then centrifuge the cells into a pellet.
Discard the pellet and collect the supernatant. This is your lysate.
Determine the total protein concentration of your sample lysate by
testing a small portion of the lysate with a commercially available protein
quantitation assay such as the BCA.
This will assist you in loading equal amounts of protein into your gel.
Western Blots are traditionally preformed under reduced and denatured
conditions.
These conditions will allow proteins to be separate by their molecular weight
rather than their native conformational shape or charge.
To reduce and denature samples,
dilute each in a loading buffer such as the traditional laemmli buffer.
This buffer contains beta-mercaptoethanol, or DTT, to reduce disulfide
ridges between cysteines,
SDS to assist denaturing a provide a net negative protein,
glycerol to allow the samples to sink into each well,
bromophenol blue to visualize the lysate
and an iconic buffer.
Votex each sample and incubate at 95 degrees Celsius for five minutes to
completely denature the proteins. You are now ready to load your samples into an
SDS page gel.