GAPDH, Tubulin, Actin, how should I choose?

We all know that there are many uncertain factors in the Western blot experiment, so the rigorous design of the Western blot experiment requires a good reference system, such as Marker, positive control, internal reference and so on. Today we will focus on the internal parameters of the WB experiment.

1.What is the internal reference? What are the functions of internal reference?

Internal reference is internal reference (Internal Control). For mammalian cells, proteins (Housekeeping Proteins) are generally encoded and expressed by butler genes. Because they are encoded and expressed by butler genes, their expression in various tissues and cells is relatively constant. Therefore, it is commonly used as a reference to detect changes in protein expression levels. The experimental operation of WB involves protein extraction, protein quantification and denaturation, protein loading and electrophoresis, transfer of protein from gel to membrane, membrane blocking, primary and secondary antibody incubation, and substrate exposure for color development. In addition, the internal reference detection is also required to compare the errors in the WB operation process to ensure the accuracy and authenticity of the results.

2.What are the common internal Control?

There are many common internal controls in mammals. In whole cells or cytoplasm, the common internal controls are Tubulin, actin, GAPDH, etc.; in the nucleus, the common internal controls are Lamin B1, HDAC , PCNA, Histone H3, etc.; in mitochondria, the common internal controls are HSP60, COX IV, etc. The following focuses on three common internal controls in whole cells or cytoplasm:

GAPDH: GAPDH or G3PDH is an acronym for glyceraldehyde-3-phosphate dehydrogenase. GAPDH is an enzyme in the glycolysis reaction and is widely distributed in cells in various tissues. The enzyme gene is a housekeeping gene, which is expressed at a high level in almost all tissues. The protein expression level in the same cell or tissue is generally constant, and it is not subject to inducing substances such as some recognition sites. Therefore, it is widely used as a standardized internal reference for experimental operations such as Western blot;

β-Actin：β-actin is composed of 375amino acids and its molecular weight is about 42-43kDa. As an internal reference, β-actin is recognized as an internal reference, which is for most tissues and cells. it is widely distributed in the cytoplasm and is very rich in expression, accounting for 50% of the total protein of all cells. However, in some special cases, such as adipose tissue or cells, the expression of β-Actin is very low.

β-Tubulin：β-Tubulin, or tubulin, is a cytoskeletal protein of cells. Tubulin can be divided into many kinds of tubulin, such as α, β, γ, δ, ε and so on. Among them, α-Tubulin and β-Tubulin can form heterodimers, which are the two most important Tubulin for microtubule formation. The molecular weights of α-tubulin and β-tubulin are 55kDa and 50kDa, respectively, and the actual detection bands are about 55kDa.

3.So what's the difference between these three internal references?

GAPDH is a metabolic protein and its expression is relatively constant in living tissues. However, when making samples after induction or detecting modified antibodies such as phosphorylation, the expression of GAPDH may be affected by induction treatment. While Actin and Tubulin are structural proteins, there are differences in cellular structure in different tissues. For example, the expression of Tubulin in brain tissue is higher (because the axons and dendrites of nerve cells are maintained by Tubulin), while the expression of Actin may be lower. For example, the special functions of skeletal muscle, heart muscle and smooth muscle lead to changes in the expression of Tubulin. The expression of β-Actin in myocardium may be decreased, while the expression of α-Actin may be high.

4.How to choose the appropriate internal reference?

The selection of internal reference mainly considers the following four factors:

(1) Species source

Due to the different species and sources of the samples, the selection of internal reference is different. For mammalian samples, proteins stably expressed in mammals need to be selected as internal reference; similarly, for plant samples, proteins stably expressed in plants need to be selected as internal reference;

(2) Cell localization

The cellular localization of the target protein is different, and the suitable internal reference is also different. For example, β-actin, Tubulin, etc. can be selected for whole cell lysate, Na+/K+-ATPase is generally selected for membrane protein, and Lamin B1, Histone H3, etc. are selected for nuclear protein;

(3) Molecular weight

The molecular weight of the internal reference protein needs to be different from the target protein, otherwise the two bands cannot be separated. The selected internal reference protein should have a different molecular weight (>5 KDa) from the target protein in order to distinguish the target protein bands.

(4) Expression factor

The selection of internal parameters also needs to consider the actual experimental environment. GAPDH is a metabolic protein, which is stably expressed in living tissues. β-Actin and β-Tubulin are structural proteins, and the cellular structures of different tissues are different. In tissue hypoxia, diabetes and other factors will lead to increased expression of GAPDH, in this case GAPDH is not suitable to be an internal reference.

I have introduced so many choices of internal reference protein. Are there any related product recommendations? Here we recommend an internal reference cocktail, which is specially designed for WB experiments and provides a real internal reference control product solution. This cocktail kit contains:

• ① The three most commonly used internal reference antibodies, classic GAPDH, β-Actin and β-Tubulin, which are cited in hundreds of references, can be used for common species samples including human, mouse, rat, etc., and are widely used in different Internal reference control for sample type and molecular weight;
• ② Ready-to-use WB positive control, which can be directly loaded to provide a real WB experimental reference;
• ③ SuperKine™ Enhanced Antibody Diluent, enhances the WB target band signal, eliminates background interference, and allows WB experiments are simpler.

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