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.