Name: 西方印跡（WB）可視化協議 (Western Blot (WB) Visual Protocol)
Uploaded: 2021-01-14T06:37:33.000Z
Duration: 14 min 37 s
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Welcome to the Novus Visual Protocol Series.

In this video we will learn how to perform all phases of a Western Blot using the

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.

we wash the cells twice with ice cold PBS

and enough lysis buffer to cover the cells.

The choice of lysis buffer depends largely on your

We scrape the cells and transfer the cell solution on a centrifuge tube

In order to solubilize membrane bound proteins, we will require stronger

extraction detergents compared to isolated cytoplasmic proteins.

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 30 minutes.

Discard the pellet and collect the supernatant.  This is your lysate.

Determine the total concentration of your protein lysate by

with a commercially available protein quantitation assay

This will assist you in loading equal amounts of protein into your gel.

Western Blots are typically preformed under reduced and denatured

conditions.  These conditions will allow proteins to be separate by their

rather than their native conformational shape or charge.

dilute each in a loading buffer such as the traditional laemmli buffer.

This buffer contains beta-mercaptoethanol, or DTT, to reduce disulfide ridges

SDS to assist denaturing 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 at 95 degrees Celsius for five minutes to

completely denature the proteins.  You are now ready to load your samples

For this next step we will separate the individual proteins in our sample

using a positive electrode to attract a negatively charged protein.

we load our previously prepared protein samples into a commercially available

Gels are available in fixed percentages or gradients of acrylamide.

The higher the acrylamide the smaller the proportion of gel

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

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

10-30 micrograms of total protein is a suggested starting point

as well as the entire amount of sample loaded.

You will also need to reserve at least one well for prestained molecular weight

The ladder will allow you to monitor protein separation during

electrophoresis and subsequently verify protein weight in your sample during

Close the electrophoresis unit and connect it to a power supply.  Most units

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

In this next step, we will transfer our separate proteins out of the gel

This is based upon the same principal as the previous step

in which an electric field is charged to remove the negative proteins

Transfer can occur under wet or semi-dry conditions.

Here we will demonstrate the traditional wet transfer method.  Start by removing

cutting the top portion containing the wells.

Notch the top left corner to indicated gel orientation.

Float the gel in transfer buffer while preparing the transfer sandwich.

and your choice of either PVDF or nitrocellulous membrane.

PVDF must first be activated by soaking the membrane in ethanol for

two minutes.  But other than this the PVDF or choice of nitrocellulous

Notch the top left corner to indicate blot orientation

and incubate membranes in transfer buffer for 10 minutes.

Create a stack by placing the following components

from the black negative cathode to red positive anode:

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西方印跡（WB）可視化協議 (Western Blot (WB) Visual Protocol)

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