Why need internal controls for Western Blot?

Western Blot (WB), one of the most routine experimental methods for biological laboratories, is almost an experimental tool that every biology experimenter should master. WB results can be used to qualitatively analyze the expression product, but also can indicate the trend of the target protein. For many experimenters, sometimes Western Blot is very simple to do when everything goes well. But it is often the case that c there will be a variety of problems: no strip, false positive, multiple bands, the expression trend does not meet expectations, and so on. To solve the problem, researchers has to review the process from the very beginning: the antibody, sample preparation, transfer, electrophoresis and other steps. The process is often very haggard.

After all, as a biological active macromolecule, the reaction between antibodies and antigens is not as clear as 1 + 1. The use of this uncertain reagent to determine the same little known expression products, is indeed dubious. Therefore, the rigorous WB experimental design requires a good reference system, which is very useful for the experimental results analysis. Especially when facing problems in your experiments, it is easy to find out the problem with the reference system, without having a clue. Good reference systems typically include molecular weight markers, blank controls, positive controls of known standard products, and loading controls.

Loading control is the most easily overlooked item. We could use WB to compare the target protein expression in different conditions or different tissues. The basis for comparison is to load same amount of cell or tissue samples. Especially, when the expression is not high, the difference in sample size is likely to affect the analysis of the results. So, you need loading control. Internal control is the internal control, for mammalian cells generally refers to the gene encoded by the housekeeping gene protein. Their expression in each tissue and cells is relatively constant and it is often used in the detection of protein expression changes. In the WB experiment, in addition to protein extraction, protein quantification, equivalent protein loading electrophoresis, transfer film, target protein antibody incubation, color and other steps, it is also necessary to use loading controls to adjust the process to ensure the accuracy of the experimental results.

Analysis of the experimental results is very simple: if the amount of protein sample is limited, and only enough for one electrophoretic transfer membrane experiment. The quantity of loading controls and target proteins are detected respectively. The target protein content of each sample was divided by the quantity of loading controls, and the obtained value was the relative content of the target protein in each sample after loading controls adjustment. Use this value to compare and analyze between the samples and get target protein content differences between samples. If the amount of sample is sufficient, you can first check the loading controls, observe whether color band is consistent between samples. Then you can adjust the sample loading quantity of Western Blotting according to the results until the quantity of loading control is consistent. If the quantity if consistent, you can analyze protein expression changes between different samples.

Commonly used proteins are GAPDH and cytoskeletal beta-actin or beta-tubulin (known as housekeeping protein, HKP). Recent reports showed that GAPDH is more frequently used. GAPDH is a key enzyme involved in glycolysis, consisting of four subunits of 30-40 kDa and a molecular weight of 146 kDa. The GAPDH gene is highly expressed in almost all tissues and is widely used as a loading control for Western blot. The method is to divide protein content by the GAPDH content of the sample to obtain the relative content of the target protein. Then you can use the relative content of the target protein for comparison.

However, there are some practical problems with housekeeping proteins as WB loading controls: First, detecting low abundance of target protein requires a large quantity of sample loading. As housekeeping proteins are mostly high abundance of protein, so HKP antibody signal may exceed the detection of the dynamic linear range. Thus, sometimes it cannot reflect the difference of the quantity of sample or other parameters. In other words, in the case of HKP signal saturation, it cannot still reveal the target actual protein expression changes. Second, under some experimental conditions and systems, HKP levels is not constant, such as different treatment conditions and sources of the sample or at various stages of development. The effective way to solve these problems is to normalize the total protein as the internal control. At present, more and more experimenter began to use the total protein as the internal controls to get more accurate and stable results, and enhance the credibility of the data. The total protein is wider than the linear detection range of HKP (as shown in Figure A), which is more stable throughout the experiment and system (as shown in Figure B), thus effectively eliminate the experimental conditions and the impact of the system on the results and reflect changes in target protein expression more accurately.