Total protein normalization is the preferred method when it comes to normalizing Western blot protein signals, as it simplifies the quantitation workflow and eliminates the need for a housekeeping protein to normalize against. This method also avoids situations where the housekeeping protein expression changes between samples or is expressed in levels outside the dynamic linear range of your protein of interest. You also eliminate the need to strip and re-probe the membrane if your housekeeping gene migrates too close to your protein of interest.
Stain-Free™ gel technology simplifies the process further by removing the need for detecting total protein amounts after protein separation with a colorimetric or fluorescent stain. Stain-Free gels (Bio-Rad) contain a trihalo compound that crosslinks with tryptophan residues in proteins, producing a fluorescent signal under UV light. The signal can be detected right after separation in the gel and after transfer to a membrane.
In this application note, we demonstrate how to combine the ease-of-use of FluorChem™ imaging systems with StainFree gels to normalize the chemiluminescent signal from Western blots using AlphaView® software.
