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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

  • 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 on your

  • choice of 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 30 minutes.

  • Then centrifuge the cells into a pellet.

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

  • Determine the total concentration of your protein lysate by

  • testing a small portion of your 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 typically 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 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

  • into an SDS page gel.

  • 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 a negatively charged protein.

  • 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 the smaller the proportion of gel

  • percentage.

  • 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 the entire 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.

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

  • and into a solid membrane or blot.

  • This is based upon the same principal as the previous step

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

  • towards a positive electrode.

  • Transfer can occur under wet or semi-dry conditions.

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

  • the gel from its cassette

  • 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.

  • To make the transfer sandwich

  • you will need a cassette,

  • sponge, filter paper

  • the gel

  • 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

  • membrane is a personal preference.

  • 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:

  • sponge,

  • filter paper,

  • membrane,

  • (Be careful not to touch the gel or membrane with your bare hands and use

  • clean tweezers or spatula instead.

  • Touching the membrane during any phase can contaminate the blot and lead to

  • excessive background signal. ),

  • filter paper

  • and sponge.

  • Use a clean roller with each layer to gently roll out any bubbles that may be

  • present

  • since bubbles will inhibit efficient protein transfer.

  • Lock the cassette and place it in the apparatus containing cold

  • transfer buffer

  • ensuring that the cassette is properly positioned from negative to positive.

  • In order to prevent heat buildup, it is beneficial to transfer with a cold

  • pack in the apparatus

  • apparatus or in a cold room with the spinner bar placed at the bottom of the chamber.

  • Close the chamber and connect to a power supply.

  • Perform the transfer according to the manufacturer's instructions which is

  • normally 100 volts for thirty to one hundred and twenty minutes.

  • After electrotransfer of our proteins to a membrane, we will

  • now block the blot,

  • apply a primary antibody specific for our protein of interest and then a secondary

  • antibody which will recognize the primary antibody.

  • Start by removing the membrane from the cassette and rinsing three times in water.

  • As an optional step, we can verify the proteins were transferred successfully

  • by staining the membrane with ponceau red.

  • Incubate the membrane in ponceau for five minutes and wash with water until

  • the bands are clear.

  • After verification

  • the blot can then be de-stained by continuing to wash with water

  • or TBS twine

  • until the dye is completely removed.

  • We need to block all areas of the blot which do not contain protein.

  • This will prevent non-specific binding of the antibody and reduce overall

  • background signal.

  • Common blocking buffers include 5% non-fat dry milk for the assay

  • in a TBS-Tween solution.

  • However do not use a milk solution when probing with phosphor-specific antibodies

  • as it can cause high background from its endogenous phosphoprotein, casein

  • Incubate the membrane with blocking solution for one hour at room

  • temperature

  • under slight agitation.

  • Decant the blocking solution and wash with TBS twine for five minutes.

  • We are now ready to add our antibody. Dilute the primary antibody in a

  • blocking buffer at the concentration recommended on the datasheet.

  • Incubate overnight at 4 degrees Celsius with gentle shaking.

  • A recommended optional step is to also use a positive control antibody

  • which allows the user to verify equal amounts of total protein were loaded into

  • each well and aides in troubleshooting by removing any

  • uncertainties with the Western Blot procedure.

  • The next day,

  • decant off the primary antibody and wash the membrane with large volumes

  • of TBS twine and vigorous agitation

  • five times for five minutes each.

  • These stringent washes are extremely important for removing non-specific

  • background signals.

  • After washing, dilute the secondary antibody in blocking solution and incubate

  • the membrane for one hour at room temperature at the concentration

  • recommended on the datasheet.

  • In our example the secondary is also conjugated to HRP for later

  • detection.

  • Decant membrane and wash secondary with large volumes of TBS twine with

  • vigorous agitation five times for five minutes each.

  • You are now ready for the detection phase.

  • In this final phase, we will demonstrate signal development using the most common,

  • most sensitive and most inexpensive detection method the electrochemiluminescence

  • (or ECL) reaction.

  • This method utilizes the HRP enzyme,

  • which was conjugated to the secondary to catalyze the ECL reaction and produce

  • light.