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 already 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 loaded 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.