Subtitles section Play video Print subtitles 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 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.