Western blotting is one of the most ubiquitous procedures used in the life science research lab today. Commonly used to assess relative protein expression differences, accurate Western blot quantitation requires careful attention to procedural details and normalization.
The most common normalization method is the use of housekeeping proteins (HKPs). This article will review the frequent challenges of using HKPs for normalization and propose an alternate method for verifying HKP results, and/or possibly replacing HKP normalization.
Tubulin, GAPDH, and actin are frequently chosen as HKPs due to their general lack of variability associated with changes in experimental conditions. As a number of publications have shown, however, caution should be used when selecting HKPs since not all HKPs are constant under changes in experimental conditions or sample types. For this reason, it is often recommended that results are validated using multiple HKPs, adding time and complexity to Western blot experiments.
The process of using HKPs for blot normalization can be daunting. Two widespread techniques are “strip and re-probe” and multiplex fluorescent detection.
HKP Normalization Methods
Using the strip and re-probe method, the protein of interest is probed and detected. Antibodies and detection chemistries are then stripped from the membrane using some combination of heat, detergent, or reducing agent. The blot may then be re-probed with HKP specific antibody and re-detected. Not only is the process time consuming, but inevitably, the stripping process will remove some level of antigen, thereby compromising downstream results.
Multiplex fluorescent Western blotting is a more elegant solution, whereby multiple antigens can be simultaneously probed and detected using multiple fluorescently labeled secondary antibodies. Western blotting frequently requires optimization of blocking reagents, antibody concentrations, and incubation times. Users need to be mindful of challenges like antibody cross-reactivity, and should have an optimization process in place that validates the detection of each antigen separately, before attempting a multiplex detection.
The challenges associated with the use of HKPs by stripping and re-probing, or optimization of multiplex fluorescent blot detection, can be avoided. A new, easier method uses Stain-Free technology for total protein Western blot normalization.
Benefits of Stain-Free Technology
Stain-Free technology is a unique in-gel chemistry that is available in Bio-Rad TGX Stain-Free precast gels. The gel formulation incorporates a trihalocompound that when exposed to UV irradiation activates a covalent reaction between the trihalocompound and tryptophan residues on the proteins in the gel, resulting in UV induced fluorescence.
Stain-Free technology enables fluorescent visualization of 1-D SDS PAGE gels and corresponding blots using Bio-Rad Laboratories’ (www.biorad.com) ChemiDoc MP imaging system (Figure 1A). The quality of SDS-PAGE separations before blotting can be easily monitored and the transfer efficiency of the blotting process can be quickly inspected by imaging both the membrane (Figure 1B) and the SDS gel after the blotting process. Furthermore, using Image Lab 4.0 software, which is included with the ChemiDoc MP, the relative amount of total protein in each lane on the blot can be calculated and used for quantitation normalization.
Stain-Free technology offers comparable sensitivity to conventional blot stains such as SYPRO Ruby and Ponceau S, and provides better reproducibility and linearity. Linear range of the Stain-Free technology is up to 80 µg protein for 18-well and up to 110 µg per lane for 12-well Criterion mid-size gels. This range fits well with usual protein loads in quantitative Western blotting experiments and enables loading control calculations over a wide protein-loading range.
HKP-based normalization with GAPDH, actin, or tubulin needs to be optimized for antibody dilutions, incubation times, and imaging settings. This is a very lengthy process compared to Stain-Free technology, which gives accurate, standardized protein-loading control with no optimization.