Control antibodies, as the name means, are a series of internal control antibodies. Generally speaking, control antibody can be divided into two groups, including isotype control antibodies and loading control antibodies.
Isotype control antibodies refer to a group of primary antibodies that lack specificity to the target, but match the class and type of the primary antibody used in the application, especially in flow cytometry experiments. Isotype control antibodies are used as negative controls to help distinguish non-specific background signal from specific antibody signal.
Loading control antibodies are known to be constitutively and stably expressed at high levels in almost all tissues and cells. Loading control proteins, provide a reference to evaluate the changes in expression levels of target proteins and help to ensure that any increase or decrease in target protein levels is due to your experimental manipulations, and not due to errors in the Western blot procedure. Housekeeping proteins, such as GAPDH, tubulin, and actin, represent some of the well-known loading control proteins.
CUSABIO offers a wide selection of monoclonal loading control antibodies for GAPDH, α-tubulin, and β-actin for several species. HRP- and biotin-conjugated versions of these antibodies are also available. In this article, we primarily focus on loading control antibodies and list several questions about CUSABIO loading control antibodies. Additionally, in the last section, we introduce five common loading control proteins and antibodies.
1. What is the purpose of loading control antibodies in Western blot?
Loading control antibodies are used in Western blotting for two main purposes:
- To estimate relative amounts of total protein loaded in each lane. (This information is needed to determine if the difference in levels of the protein of interest in different samples is due to loading and transfer errors or to real changes in protein expression.)
- To troubleshoot the Western blotting procedure.
2. What are the important factor when you choose the suitable loading control antibodies in experiment?
As mentioned earlier, loading control antibodies are reference objects that can monitor whether the whole test system is normal, such as protein extraction process, membrane transfer system, color development system, etc. They can also be used to compare the expression of target protein in different cell tissues under different conditions. When choosing a right loading control antibody as an internal reference, you need to pay attention to the following aspects.
- Sample Source: in mammalian tissue or cell, it is mainly represented by Actin, Tubulin, GAPDH, Lamin B1, PCNA. But if it is a plant sample, it is mainly represented by actin and GAPDH.
- Target Protein Molecular Weight: common loading control antibodies involve β Actin, β Tubulin and GAPDH, the molecular weights of them are 42 kDa, 50 kDa and 37 kDa, respectively. The molecular weight of the target protein is preferably more than 5kd from the molecular weight of the internal reference protein, so as to separate the target protein and the internal reference.
- Target Protein Expression Site: Housekeeping proteins are differ in different expression site. For instance, Tubulin, β Actin and GAPDH are whole cell proteins, the locality of PCNA is cell nuclear, and Lamin B1 is located in nuclear membrane, and so on. As the table 1 shows, we summarize the characteristics of common housekeeping proteins, including name, type and molecular weight.
Table 1. The characteristics of common housekeeping proteins
||Plant Whole cell
3. Why choose CUSABIO loading control antibodies?
Whatever your requirements, we have a loading control antibody to suit your experiment.
- 100% Risk-free Performance Guarantee
CUSABIO guarantees its antibodies to work in the applications as well as react with the species listed in our website or product data sheets for a period of 12 months from the manufacturing date. We stand behind all our products and are confident that they will perform as designed. If one of our antibodies does not perform as described on the data sheets, our technical team will work with you to troubleshoot the issue. If this troubleshooting process could not solve your problem, we will be happy to replace it or issue a refund for the antibody purchase price.
- Constant Supply and Prompt Delivery
As an original manufacturer, CUSABIO generates and validates all antibodies in-house, so we can promise the constant supply, prompt delivery as well as quality guarantee for all antibodies we have.
- High performance
All of our loading control antibodies are monoclonal antibodies, and have high sensitivity and reproducible results.
We provide two pack sizes of 50 µg and 100 µg. And the most important point is that all of the loading control antibodies are in stock.
4. Five common loading control antibodies
In this section, we list the top of five common loading control antibodies from CUSABIO.
Actin, an important cytoskeleton protein of cells, can be roughly divided into six types. Among of them, four types of actin are specific for different muscle tissues, and the other two are widely distributed in various tissues, including β-actin and γ-non-muscle actin. And β-actin is recognized as an internal reference for most tissues and cells.
Beta-actin is widely distributed in the cytoplasm and is abundantly expressed in 50% of the total protein of all cells. However, in a few special cases, such as adipose tissue or intracellular, the expression of β-Actin is very small. It is composed of 375 amino acids with a molecular weight of about 42-43 kDa. It is suitable for the detection of 38kD-48kD size target protein in WB experiments. But, in adipose tissue, β-Actin expression is very low, not suitable as an internal reference.
GAPDH, also known as glyceraldehyde-3-phosphate dehydrogenase, is a key enzyme involved in glycolysis. It consists of four subunits (each subunit is 30-40 kDa), and its molecular weight is about 146 kDa with a detection band of approximately 36 kDa. The GAPDH gene is expressed at high levels in almost all tissues and can be widely used as an internal reference for Western blot protein standardization. But note that it is not suitable for the detection of 30kD-40kD size target protein in WB experiments. Moreover, in some cells, since tissue hypoxia, diabetes and other factors will lead to increased expression of GAPDH, it is also not suitable for internal reference.
4.3 β-Tubulin loading control antibody
Tubulin, like the actin, also is a cytoskeleton protein of cells. Tubulin can be divided into various tubulins such as α, β, γ, δ, ε, etc. Among of them, α-Tubulin and β-Tubulin can form heterodimers, which are the two main types of Tubulin which form microtubules. The molecular weights of α-tubulin and β-tubulin are 55kDa and 50kDa, respectively, and its actual detection bands were around 55kDa.
As an internal reference, the protein level of β-tubulin is usually not changed, so it is more widely used for the reference of whether the sample loading is consistent in Western Blotting, and is often used for immuno-staining to observe the microtubule structure of cells. However, be note, it is not suitable for WB experiments for detecting target proteins of 50kD-60kD size. Additionally, in adipose tissue or cells, β- Tubulin is rarely expressed and is not suitable for internal reference.
Proliferating Cell Nuclear Antigen, also abbreviated as PCNA, was first discovered and named by Miyachi in the serum of patients with SLE (systemic lupus erythematosus) in 1978, and was named for its presence only in normal proliferating cells and tumor cells. PCNA is a protein with a molecular weight of 36kDa, which is synthesized in the nucleus and is present in the nucleus and is an accessory protein of DNA polymerase. Because of its stable expression in the nucleus, its antibodies are widely used in internal reference antibodies of nuclear proteins.
In addition to Histone H3 and PCNA, other common nuclear protein internal references include K70, K80, Lamin A and B. In some studies, Erk2, TATA binding protein (TBP), c-Jun, c-Fos, etc. Both are used. However, be note, in addition to considering the molecular weight of the target protein, the selection of the nuclear protein internal reference needs to consider the actual experimental environment. For example, in the experiments involving cell proliferation, c-Jun is not suitable for internal reference due to changes in its own expression. During this kind of experiment, TBP, Lamin, etc. are also not suitable as internal parameters.
4.5 COX IV loading control antibody
Cytochrome c oxidase (COX), an enzyme complex of the mitochondrial respiratory chain, is a heterologous oligomeric enzyme that usually contains 13 subunits located in the mitochondrial inner membrane. The three largest subunits that form the catalytic center in cytochrome c oxidase are encoded by mitochondrial DNA, and the remaining 10 subunits, including COX IV, are encoded by genomic DNA within the nucleus. Defects in cytochrome c oxidase (COX) activity are associated with a variety of human diseases. The COX IV antibody can be used as an internal reference antibody for mitochondrial protein loading, and the target protein size is about 16 kDa.