Sample preparation, loading, uneven transfer and transfer efficiency can all affect recovery and detection of proteins on the membrane. When evaluating the amount of protein of interest on the blot, it is vital to use a normalization control. Traditionally, detection of a housekeeping gene is used for normalization to control for inconsistencies. However, housekeeping genes have specific disadvantages as normalization controls. Total protein normalization is another method for normalizing protein expression. Total protein normalization can be performed using different dyes and stains, as well as a newer stain-free technology. This guide reviews several different methods of total protein normalization.
Housekeeping genes as normalization controls
To perform normalization using a housekeeping gene, the blot is probed with antibodies to detect the protein of interest while another set of antibodies is used to detect a separate protein used as a normalization control. For quantitative studies, the ratio of the abundance of the protein of interest to the normalization control is used to quantify the amount of the protein of interest in each sample. For qualitative studies, the loading control is often presented in an image for visual comparison.
Housekeeping genes, such as such as glyceraldehyde 3-phosphate dehydrogenase (GADPH), beta-actin or tubulin, are commonly used for loading controls. These proteins are usually expressed constitutively at high levels due to their role in cell viability. However, several factors limit their utility as normalization controls.
- Studies have shown that housekeeping gene expression can change with different experimental conditions and differ between cell types and during different developmental phases (Liu, 2006; Moskowitz, 1995; Nahlik, 2003).
- The high level of expression of housekeeping genes often makes it difficult to detect a lower expressing protein on the same blot. The amount of protein required per lane to detect the protein of interest often results in a signal that far exceeds the linear dynamic range when detecting a housekeeping gene.
- For true normalization, the protein of interest and the normalization control need to be detected on the same blot. Unless the two proteins are sufficiently separated by gel electrophoresis and the membrane can be cut and incubated for each protein separately, many researchers rely on stripping and reprobing, which is not quantitative.
- Expression of the protein of interest is only compared to one other protein.
Total Protein Normalization
Total protein normalization (TPN) is a technique that can be used to quantify the abundance of the protein of interest without relying on housekeeping genes. Traditionally, TPN is performed by incubating the membrane with a total protein stain, either before or after detection with antibodies. The abundance of the protein of interest is normalized to the total amount of protein in each lane, removing variations associated with comparing abundance to a single protein. TPN is also more compatible with detecting proteins of lower abundance.
Normalization with total protein stains can be complex and time-consuming. While some stains can be used prior to immunodetection, others are incompatible with downstream antibody detection. In addition, some stains fade quickly making it difficult to document the results. A newer technique, called the stain-free approach, increases the sensitivity and reduces the complexity while saving time.