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How to Choose an Loading Control Antibody?
Loading control, for mammalian cell expression, generally refers to proteins encoded and expressed by housekeeping genes, whose expression is relatively constant in various tissues and cells, and which is commonly used as a control when detecting changes in protein expression levels.
The detection of internal control is crucial in Western Blot experiments, which is used to correct the experimental errors during protein quantification and sample preparation, and to ensure the accuracy of the experimental results. In addition, the use of internal reference can be used as a blank control to detect whether the protein transmembrane situation is complete, whether the entire Western Blot color development or luminescence system is normal.
So, how should we choose the loading control antibody? Let’s take a look at the commonly used Loading control.
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Common Loading Control
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Introduction and Application Scenarios
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CUSABIO Loading Control Antibody
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GAPDH
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GAPDH gene is expressed at high levels in almost all tissues, and it can be widely used as an internal reference for protein standardization in Western Blot.
※It is not suitable for use in WB experiments that detect target proteins of 30kDa-40kDa size. In some cells, the expression of GAPDH is increased due to tissue hypoxia, diabetes and other factors, also unsuitable for internal reference.
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GAPDH Monoclonal Antibody
CSB-MA000071M0m
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β-Actin
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β-actin is widely distributed in the cell plasma, and its expression is very abundant, and its content accounts for 50% of all total cellular proteins. β-actin consists of 375 amino acids, and its molecular weight size is about 42-43kDa.
※It is suitable for use in WB experiments to detect target proteins of 38kDa-48kDa size. In adipose tissue, the expression of β-Actin is very low and is not suitable for use as an internal reference.
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Beta-Actin Monoclonal Antibody
CSB-MA000091M0m
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β-Tubulin
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β-tubulin is widely used as an internal reference for the consistency of the sample volume in Western Blotting, and for immunostaining to observe microtubule structure of cells.
※It is not suitable for use in WB experiments that detect target proteins of 50kDa-60kDa size. The expression of β-Actin in adipose tissue or cells is very small and is not suitable for internal reference.
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TUBB Monoclonal Antibody
CSB-MA025318A0m
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Please note that the internal reference antibody selection needs to consider the actual application scenarios, such as:
- When doing multi-tissue and multi-cell samples to compare the expression, it is better to choose GAPDH as the internal reference, because GAPDH is a metabolic protein, and its expression is relatively constant in living tissues. And β-Actin and β-Tubulin are structural proteins, and there will be variability in the cell structure of different tissues.
- When doing post-induction samples or detecting modified antibodies such as phosphorylation, structural proteins should be chosen as internal references, such as β-Actin and β-Tubulin.
- When designing the experimental program, you should consider the actual experimental factors and consult the corresponding literature, and also pay attention to whether the expression of the internal reference is abnormal or not during the experimental process.
- When doing Western Blot, the intensity of the bands of the internal reference should also be controlled appropriately. Do not overexpose the bands.
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Of course, a better way is to test a small amount first
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Kit
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Protein
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Antibody
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Molecular Biology
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Postal Address:
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Website: www.cusabio.com
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