b'Post-transferOnce the protein transfer process is completed, theMonitoring transfer efficiencymembrane is removed from the transfer sandwich and is ready for any post-transfer membrane treatment.Transfer efficiency can vary dramatically among proteins, Before committing valuable time and detection reagentsbased upon the ability of a protein to migrate out of the to western blot processing and detection, it is prudentgel and its propensity to bind to the membrane under to assess the efficiency of the transfer step. Signala particular set of conditions. The efficiency of transfer enhancers may be used at this stage, before thedepends on factors such as the composition of the gel, membrane is incubated with blocking buffer, if there is acontact of the gel with the membrane, the position of the need for increased sensitivity. electrodes, transfer time, field strength, and the presence of detergents, as well as the size and composition of the protein of interest.Some researchers may use prestained protein ladders to assess transfer efficiency. Others may stain the gel to confirm that proteins have migrated out of the gel. However, these methods are unreliable because they do not necessarily reveal how effectively proteins have transferred to the membrane. A better method to monitor transfer efficiency relies on staining the membrane for total protein with a dye such as Ponceau S or amido black 10B. Because dyes may interfere with antibody binding and detection, a protein stain that is easily removable is ideal. Ponceau S stain is the most widely used reagent for reversibly staining proteins on a membrane, although it has limited sensitivity, does not photograph well, and fades quickly, making documentation difficult. Superior alternatives for staining protein on nitrocellulose or PVDF membranes are available that are easily photographed and do not fade until removed. These dyes also allow the detection of low-nanogram levels of protein on membranes. No-Stain Protein Labeling Reagent (page 84) can also be used for total protein labeling after gel transfer. It labels proteins covalently and can be used to normalize for protein sample loading variations when performing quantitative western blotting experiments.25'