b'Normalization and quantitation in western blot detectionAs the amount of protein loaded on an electrophoresisTotal protein normalization (TPN) is a better alternative gel can vary greatly from sample to sample, leadingto using housekeeping proteins or total target protein journals have developed protein normalizing guidelines fornormalization for posttranslationally modified proteins. submitting quantitative western blot data.It avoids the variability and inaccuracy of using housekeeping proteins, the time-consuming effort to One common method of protein normalization reliesoptimize conditions, and the cost of the immunodetection on measuring the abundance of a stably expressedreagents to detect the normalization targets.housekeeping protein that is endogenous to all the samples in an experiment. Proteins such as -tubulin,For TPN, the total protein on a blot is labeled or stained -actin, or glyceraldehyde 3-phosphate dehydrogenaseto enable the relative amount of protein loaded in each (GAPDH) are commonly used as loading controls forlane to be compared and normalized to a reference protein normalization, but there are potential drawbacks,lane. Ponceau S and several reversible fluorescent dyes as the expression of housekeeping proteins can vary withare available to stain the total protein on a blot for TPN. experimental conditions and they often have saturatedA shortcoming of these dyes is that they must be removed western blotting signals. These drawbacks can bebefore the western blotting antibody detection steps, minimized with optimization, but this optimization can berequiring the blots to be imaged after staining, followed by time-consuming. One must first confirm that the signalremoval of the dye before moving to the immunoblotting obtained for the housekeeping protein is linear oversteps. The finished western blot must be imaged again at a wide concentration range, such that it can be usedthe end of the process before moving on to normalization as a reliable reference for normalization, and that it isof the results. These staining, destaining, and multiple unaffected by the conditions of the experiment. The useimaging steps are time-consuming.of antibodies to detect housekeeping proteins may require the extra steps of stripping and reprobing the blot toOther than using blot stains like Ponceau S for TPN, an detect the experimental targets, which adds another layeralternative method has become available. However, this of time investment. method requires the purchase and use of a specialized gel that may yield very different results from your current An alternative protein normalization method is total targetoptimized system for separating your target proteins. protein normalization for posttranslationally modifiedThe method requires a specific gel imager to capture proteins. Targets with posttranslational modificationsthe image and requires the total protein on the blot (e.g., phosphorylation, glycosylation, acetylation,to be imaged before the immunoblotting step, as the and ubiquitination) are normalized to their respectivechemiluminescent signal from the target proteins interferes total protein using a pan antibody that recognizeswith the total protein signal on the blot.the target regardless of modification state. There are multiple drawbacks to using this method to normalize for posttranslationally modified target protein. The pan or total target protein antibody must recognize all posttranslationally modified versions of the target as well as the unmodified target. In addition, the expression of the total protein target used for normalization must be constant across all experimental conditions. Further, the linear dynamic range for this method is often narrow.67'