Principles of Western Blot Internal Reference Antibody Selection
There are many kinds of Western Blot internal reference antibodies, such as β-actin, β-tubulin, GAPDH, Lamin B, etc., so how should we do the selection for our own experiments? Below is a brief introduction to the principles that should be followed in selecting the internal reference antibody:
Species source of the sample
The first thing to consider when choosing an internal reference antibody is the source of the experimental sample.
1. mammalian tissue or cell samples, usually choose β-actin, β-tubulin, GAPDH, Lamin B, Histone H3, Na/K ATPase and so on.
2. plant actin, Rubisco, etc. for samples of plant origin.
3. For samples from other sources, which are less studied, the researcher needs to refer to the literature to select the appropriate antibody as internal reference.
Molecular weight of target protein
The molecular weight of the target protein should be taken into consideration when choosing an internal reference antibody. Usually, the molecular weight difference between the target protein and the internal reference protein should be more than 5 kDa. For example, if the molecular weight of the target protein is 45 kDa, it is not suitable to choose β-actin (molecular weight of 42 kDa) as the internal reference, but GAPDH (molecular weight of 38 kDa) or β-tubulin (molecular weight of 55 kDa) can be considered as the internal reference.
Expression site of target protein
Generally speaking, for the detection of intracytoplasmic proteins, antibodies against β-actin, β-Tubulin and GAPDH can be chosen; for the quantification of nuclear proteins, especially when the samples are derived from proteins in the nucleus of the cell, the choice of the appropriate internal reference for nuclear proteins can better reflect the value of the internal reference, and the commonly used antibodies against the internal reference for nuclear proteins are Lamin A, Lamin B, Histone, H3, and other common internal reference antibodies. H3, in addition, other common nuclear protein internal reference are PCNA, K70, K80, etc. In some literature reports, Erk2, TATA binding protein (TBP), as well as c-Jun, c-Fos, etc. have been used; for the detection of membrane proteins, the most commonly used internal reference antibody is Na/K ATPase; for the detection of mitochondrial proteins, the most commonly used antibodies are VDAC1 and COOAC1. VDAC1 and COX IV are commonly used as internal reference antibodies.
The above principles are only for the general situation, and it should be noted that the selection of internal reference also needs to take into account the actual experimental environment, such as some cells due to tissue hypoxia, diabetes and other factors will lead to increased expression of GAPDH, in which case GAPDH is not suitable for use as an internal reference; and for example, in experiments involving cell proliferation, c-Jun is not suitable as an internal reference because of the lack of oxygen and diabetes, and c-Jun is not suitable as an internal reference. For example, in cell proliferation-related experiments, c-Jun is not suitable for internal reference due to its own expression changes; and in apoptosis experiments, TBP, Lamin, etc. are also not suitable for internal reference. Therefore, when designing the experimental program, we should consider various factors and consult the relevant literature. During the experiment, we should also pay attention to the fact that if the expression of the endogenous reference is abnormal, it may be due to special reasons, so we need to consult the relevant literature and replace the endogenous reference with another antibody.