RESOURCES
The use of internal reference in Western Blotting is to test the internal reference protein with the antibody corresponding to the internal reference in the test. The internal reference is an internal reference, generally refers to the protein encoded by the housekeeping gene, and their expression in each tissue and cell is relatively constant, and is often used as a reference when detecting changes in the expression level of the protein.
Western Blotting can be used to compare the expression of target proteins in different conditions or different tissue cells when the tissue cell protein is loaded in the same amount, especially when the expression level of the target protein is not high, the difference in the amount of the sample is likely to affect the result. Analysis. Therefore, in the Western Blotting test, the internal reference test can correct the error of protein quantification and loading, and ensure the accuracy of the experimental results. In addition, the internal reference can be used as a blank control to detect whether the protein transfer film is complete and the whole Western Whether the Blotting color development system is normal. At present, Western Blotting has become a routine practice in internal and international publications.
There are many types of internal reference antibodies, such as β-Actin, β-Tubulin, GAPDH, Lamin B, PCNA, Na+/K+ATPase, etc. The following principles should be followed to select internal reference antibodies:
one. Sample species source
The first thing to consider is what species the experimental sample is derived from.
For mammalian tissues or cell samples, β-actin, β-tubulin, GAPDH, Lamin B, PCNA, Na+/K+-ATPase, etc. are usually selected.
For plant-derived experimental samples, you can choose Plant actin, Rubisco, etc.
There are few studies on other sources, so it should be reported in the literature to select the appropriate protein as an internal reference.
two. Target protein molecular weight
When selecting an internal reference antibody, the molecular weight of the target protein should be considered. It should generally be ensured that the molecular weight of the protein of interest and the internal reference protein differ by more than 5KD. For example, the molecular weight of the target protein is 45KD. At this time, β-actin (42KD) is not suitable as an internal reference. GAPDH (36KD) can be considered as an internal reference.
three. Target protein expression site
Cytoplasmic and whole-cell proteins: β-actin, GAPDH, β-Tubulin, etc.
Nuclear protein: PCNA, etc.
Nuclear membrane protein: Lamin B, etc.
Membrane protein: Na+/K+-ATPase, etc.
Mitochondrial proteins: COX IV, VDAC1, etc.
The above principles are only for the usual situation, but the problem to be noted is that the choice of internal parameters needs to consider the actual test environment. For example, in some cells, due to tissue hypoxia, diabetes and other factors, the expression of GAPDH is increased, which is not suitable for internal reference; for example, in the apoptosis experiment, Lamin is not suitable as an internal reference. Therefore, when designing the experimental scheme, these factors should be considered and the corresponding literature should be consulted. It should also be noted that the expression of the internal reference is abnormal.
