Since the first publication describing western blotting in 1979, this immunodetection technique has been widely used to alter specific proteins in complex mixtures extracted from cells or tissues. Western blotting has three basic elements: 1 separation of proteins by size, 2 transfer to solid support, 3 labeling of the protein of interest with the appropriate primary antibody, and then visualization (usually using conjugated secondary antibodies). Subsequent improvements in tools and techniques, as well as the development of highly sensitive fluorescent labels, have effectively increased the limits of detection and enabled scientists to probe tissue-specific normal and disease. However, scientists often encounter some difficulties when using quantitative western blotting to identify changes in protein levels. This is due to the fact that many proteins exhibit different expression patterns in tissues and under different physiological and pathological conditions.
While western blotting is probably the most commonly used immunological application, there are still some key technical issues that may have been overlooked for a long time. For example, does the isolation or isolation protocol affect the integrity of the protein or its post-translational modifications?Is it possible to distinguish between the explanation or aggregation of proteins and the related products of biological processesWhen the result is multiple bands, how do you determine which are the results, variations, artifacts?
Quantitative western blotting can be used to compare the relative amount of a target protein in a set of samples that can represent different individuals, conditions, disease states, or other biological variables. To accurately identify and measure total protein levels across multiple samples, scientists can use an "up-and-down control" as an internal standard. This control refers to the addition of a primary antibody against a protein that is presumed to be present in all samples and whose relative abundance is not affected by biological variation or experimental conditions. Protein targets are often good candidates for loading controls, and they are ubiquitously expressed "housekeeping" gene products. Assuming that the loading control is the same between different sample lanes, the loading control can be used to quantify the sample load in all wells to normalize the results. The use of loading controls also prevents "edge effects", which is common when using a large number of lanes, and where proteins in the outer lanes are transferred to the smear closer to the frame, resulting in more intense staining on the lanes. Loading controls can be used to show if there has been a change in the amount of protein loaded and to interpret the observed changes in the band of interest. When used correctly, loading controls ensure that proteins are correctly quantified, although there are slight differences in the amount of sample loaded across all lanes of western blotting.
Actin and -tubulin have been used as loading controls because their expression is relatively constitutive in most model systems. However, some publications have questioned the use of -actin as a standard loading control, citing differences between -actin and -tubulin level tissues and that their expression may be affected by pathological conditions. These authors recommend total protein analysis as an alternative technique in quantitative western blotting and recommend that "housekeeping" gene products should be used with caution after studying gene expression patterns in the institute. Nonetheless, -actin and -tubulin have certain advantages as internal control antibodies: they are highly conserved, have high expression levels, and exhibit good stability under most experimental conditions. It is important to note that the control must be selected based on the specific tissue or cell type being studied, and empirical testing may be required to verify the homogeneity of the loading control.
To overcome variability in western blotting and reduce errors in data interpretation, some of the following precautions may help.
An internal loading control with stable expression and minimal influence by experimental conditions was used.
Select a loading control antibody against a protein known to be expressed in the composition row of the sample.
A second loading control was used to confirm the results obtained from the first control. It can be especially valuable for new or novel samples.
The loading control should cover a wide range of molecular weights so that the control selected is similar to the molecular weight of the protein of interest. This ensures that the band of interest and the control band can be easily distinguished on the blot.
Loading control antibodies often detect large amounts of expressed housekeeping proteins, resulting in signal saturation, especially when using chemiluminescent detection methods, and oversaturation may result in the loading control band being unusable for reference and potentially hiding sample-to-sample differences in the amount of protein of interest.
References
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