Looking for a way to visualize a protein from cells or tissue samples.
Maybe using a Western blot.
Wait, wait. Come back.
Westerns are good, I promise.
My name's Meghan,
and I'll be explaining the basic steps for running a denaturing in Western Blot.
Don't let their bad reputation scare you.
Westerns are actually quite useful once you're armed with the right information.
Now to be fair, Western blots, while incredibly useful tools,
have a certain reputation and it's true, they can be tricky.
But tricky isn't impossible.
Join me in this video and I'll have you running.
Transferring and chemiluminescing like a pro!
For advanced users, Westerns can also be combined with other assays
to determine how a protein is regulated or to identify interacting partners.
Okay.
Now that we know why we might want to run a Western.
The next question is what do we need to run one?
It's quite the shopping list, so feel free to take notes.
To run a Western, you'll: need
protein samples, cell lysis buffer, BCA protein assay,
BSA standard for protein assay, loading dye, reducing agent,
an ice bucket and some ice, microcentrifuge tubes,
sds-page gel, sds-page running buffer, Powdered milk, 1X TBST,
or other wash buffer, iBlot transfer stack,
Primary antibody, a secondary antibody if applicable.
Chemiluminescent substrate, plastic wrap, clear sheet protector,
gel loading tips, ionized water, pre-stained protein standard, 96
well plate, microcentrifuge, shaker, hot plate
set to 100 degrees Celsius, Incubator set to 37 degrees Celsius, Cold rRoom,
Spectrophotometer, Electrophoresis Chamber, Power supply,
Spatula, Plastic container, Forceps,
Transfer apparatus, Gel imager.
Phew! Got everything? Great!
That brings us to the big question.
How do we run a Western?
We start by preparing our samples.
First,
will lyse cells or tissue samples on ice to release the proteins into solution.
This can be done with a number of different reagents.
So just pick the one you have on hand or the one you're most comfortable using
once the lysis step is complete.
Make sure to clear the lysates by Centrifugation to remove any cellular
debris from the samples.
Next, you'll need to determine the protein concentration of the sample.
There are many protein quantification kits available.
Most use a bovine serum albumin reference to determine
the samples concentration relative to the BSA standard curve.
Just follow the directions and you'll be fine
since you'll be calculating relative protein abundance.
It's critical
that the same amount of protein is loaded for all samples on a single block,
ensuring that each sample has the same amount of protein
instead of the same volume, but prevent ambiguous results.
Most researchers select a protein amount between ten and 50 micrograms
depending on the total volume they can load into the gel
and the amount of protein in their samples.
The exact amount used can vary from assay to assay, and that's okay.
Once you have selected the total amount of protein, you'll use
for this assay, use the concentrations calculated from the standard curve
to determine the volume of each sample that you'll need to load into the gel.
In a micro centrifuge tube combine
the required volume of sample with set amount of loading dye and reducing agent.
Some people add distilled water to bring all the samples up to the same volume.
Mix the samples by vortexing
and centrifuge briefly.
Boil the samples
at 100 degrees Celsius for 15 minutes.
This will reduce and denature the proteins in the sample.
15 minutes. Why?
That's just enough time to prep our page for loading!
SDS-page gels can be hand cast or purchased precast
from a variety of vendors.
For both gel types, remove any packaging or casting
material and combs.
Use a pipette and tip to gently rinse the wells in the running buffer,
removing the packing liquid and any air bubbles.
Gently load the gel into the electrophoresis chamber
and lock it in place.
Fill the chamber to the fill line
with the running buffer.
Using
gel loading tips, gently load a prestained protein
standard into a gel plane,
then pipette each sample into its own lane
Be very careful not to introduce bubbles when pipetting.
Attach the electrodes to the electrophoresis
chamber
and connect to the power supply.
Run the gel at a low voltage,
usually 50 to 60 volts for 30 minutes.
This allows the samples to line up and enter into the resolving gel.
After 30 minutes, increase the voltage to a final voltage of 5
to 15 volts per centimeter of gel.
The prestained protein standard will be visible on the gel as it runs.
Watch it closely and do not run your protein off the gel.
When the protein standard has separated to the desired degree,
one that will allow you to get good visualization of your protein.
Turn off power to the gel.
Remove the lid
and carefully remove the gel
from the electrophoresis chamber.
Use a metal spatula to gently separate
the sides of the gel cast.
Gently remove the gel from the cast
and transfer
to a container of distilled water.
Allow the gel
to wash for 5 minutes to remove the residual running buffer.
Note that in a wash step, the gel or membrane is fully
immersed in a liquid and is on a rocker so the liquid washes over the gel.
The next step is transferring the separated proteins from the gel
to a membrane such as PVDF or Nitrocellulose.
This is done using a transfer apparatus.
Transfers can be done
using a wet, dry,or semi dry assembly.
Each procedure is slightly different, but all involve creating a transfer
sandwich of a membrane, gel, and absorbent blotting paper,
then passing an electric current through the sandwich to migrate
the proteins from the gel to the membrane.
One quick reminder the transfer sandwich is not for consumption.
Two quick transfer tips.
If you're using PVDF to transfer, be sure to activate the membrane
with methanol before use.
For a dry transfer, you must pre equilibrate the gels
if the protein target is over 150 kiloDaltons.
To do this, soak the gel and 20%
ethanol for 10 minutes before assembling the transfer sandwich.
To assemble your transfer sandwich, make the bottom stack like so:
absorbent pad, copper anode, anode stack, and
membrane.
Place the blotting apparatus such
that the electrical contacts are aligned.
Shake excess liquid off the gel and gently
place it on top of the membrane.
Use a roller to gently remove any air
bubbles between the gel and the membrane.
Soak a piece of filter paper
deionized water.
Carefully place the filter paper on the gel
and use the roller to gently remove air
bubbles between the gel and the filter paper.
Place the top stack consisting of the cathode stack,
copper cathode,
and absorbent
pad on the filter paper.
Use a roller
to gently remove any air bubbles.
Cover the transfer apparatus
and select and run the desired program.
Once the program is complete,
carefully remove the top stack and discard.
The protein ladder should now be visible on the membrane,
indicating that the transfer was successful.
Using forceps carefully transfer the membrane to a container
of blocking buffer,
and block for one hour at room temperature.
The blocking buffer used typically milk or BSA is antibody dependent,
so take care to refer to the antibody datasheet before preparing.
All right.
You have about an hour until your gel is finished blocking.
While you wait, you can prepare your primary antibody.
You'll need your antibody diluted in blocking buffer
and a large enough amount to cover the membrane during incubation.
The concentration of the primary antibody depends on a number of factors,
such as the amount of protein loaded, abundance of the target,
and affinity of the antibody to its target.
You may need to try a range of different
concentrations before uncovering the ideal conditions.
If the ideal concentration for your conditions are unknown, try
starting with one microgram per milliliter and optimize from
there.
After the
membrane is blocked, wash three times for 5 minutes in the wash buffer.
Replacing the wash buffer for each wash.
After the third wash is complete,
carefully transfer the membrane to a container with a diluted
primary antibody.
Primary antibody incubation can be performed for 1 to 2 hours at room
temperature
or overnight at four degrees Celsius.
Generally, overnight incubation at four degrees
Celsius is recommended to reduce nonspecific background signals.
Of course,
many people also appreciate the break in this long and complicated process.
After the primary antibody incubation is complete.
Wash the membrane three times for 5 minutes in the wash buffer
just like before.
If your primary antibody is directly conjugated
to a detection label, no secondary antibody is needed.
You can go ahead and proceed with the Chemiluminescence detection step.
If your primary antibody is unconjugated,
prepare the secondary antibody during the wash step
by diluting to the desired final concentration in the blocking buffer.
The secondary antibody used
will depend on the species that the primary antibody was derived in.
If the primary antibody
was made in a mouse using the Anti-mouse secondary antibody,
if the primary antibody was made in a rabbit using the anti rabbit
secondary antibody, your antibody data sheet will have appropriate dilutions.
So use that as your guide.
Once the washes are complete,
transfer the membrane to the diluted secondary antibody solution
and incubate for one hour at room temperature.
After intubation, wash the membrane three times for 5 minutes in the wash buffer.
During the third wash, prepare the detection reagent.
The specific detection reagent used will depend on the conjugation of the secondary
antibody, but is typically horseradish peroxidase or alkaline phosphatase.
For the horseradish peroxidase detection method.
Combine a one-to-one mix of solutions A and B
on a piece of plastic wrap.
Gently remove the excess wash buffer from the membrane by tapping
and place the membrane protein side down
into the chemiluminescence substrate.
Using the forceps, gently tap the excess
chemiluminescence reagent off the membrane
and place the membrane protein
side up in a clear sheet protector.
Use a gel imager with chemical luminescent
capabilities or x ray film in a dark room for detection.
The ideal exposure time will vary depending on a variety of factors
such as protein abundance and antibody signal strength.
Start with a short exposure time of 30 to 60 seconds and optimize
from there to get the ideal image.
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