5-Step Quick Guide to Selecting Loading Control Antibody

5-Step Quick Guide to Selecting Loading Control Antibody

Loading control, typically a protein encoded by a housekeeping gene. These proteins exhibit relatively constant expression across various tissues and cells, and are commonly used as references when detecting changes in the expression levels of target proteins.

Loading control antibodies serve as the "benchmark" in experiments. They are used to calibrate differences in sample quantity and assess the stability of the experimental system. Selecting appropriate loading control antibody is a crucial step in ensuring data reliability, and is particularly vital for research on gene expression, protein quantification, and other related studies in university scientific research. How to correctly select loading control antibody? The following points can be used as a reference.

           GAPDH Rabbit Monoclonal Antibody （Cat: AMRe21263）

1. Species Origin of Samples

The first factor to consider is the species from which the experimental samples are derived. For mammalian tissue or cell samples, proteins such as β-actin, Tubulin, GAPDH, Lamin B, PCNA, and Na⁺/K⁺-ATPase are usually selected. For plant-derived experimental samples, Plant actin, Rubisco, etc., can be chosen. There is relatively little research on samples from other sources; therefore, appropriate proteins should be selected as loading control protein by referring to literature reports.

2. Molecular Weight of the Target Protein

When selecting a loading control antibody, the molecular weight of the target protein should be taken into account. Generally, it is necessary to ensure that the molecular weight difference between the target protein and the reference protein is at least 5 kDa. For example, if the molecular weight of the target protein is 40 kDa, β-actin and GAPDH are not suitable choices for loading control proteins at this time, and Tubulin can be considered as an alternative loading control protein.

3. Expression Level of the Internal Reference Protein

The selected loading control protein needs to be highly expressed in the test samples. Commonly used loading control proteins are highly expressed by housekeeping genes and are essential for cell development and the maintenance of cell viability.

4. Localization of the Target Protein

For the detection of general proteins, GAPDH, β-actin, or β-tubulin can meet the requirements. However, when it is necessary to detect subcellular organelle proteins, it is more appropriate to select loading control proteins specific to the corresponding subcellular organelles, as this better ensures the accuracy of the loading control protein.

5. Select Loading Control proteins Unaffected by Experimental Variables

a. When comparing expression levels across multiple tissue or cell samples, GAPDH is preferably used as the loading control protein. As a metabolic protein, GAPDH exhibits relatively constant expression in living tissues. In contrast, β-actin and β-Tubulin are structural proteins, and the cellular structure varies across different tissues (resulting in inconsistent expression of these structural proteins, making them unsuitable for cross-tissue/cell comparison).

b. In some cells, factors such as tissue hypoxia and diabetes can lead to increased GAPDH expression, rendering it unsuitable as a loading control protein.

c. In experiments related to cell proliferation, c-Jun is not suitable for use as a loading control protein due to its own fluctuating expression (its expression level changes with cell proliferation status).

d. During apoptosis experiments, proteins like TBP and Lamin are also unsuitable as loading control proteins (their expression is often disrupted or downregulated during the apoptotic process).

e. When working with induced samples or detecting modified antibodies (e.g., phosphorylated antibodies), structural proteins such as β-actin and β-Tubulin should be selected as loading control proteins. Their expression is less affected by induction treatments or post-translational modifications, ensuring stable reference values.

f. When working with various secretion samples (such as plasma, milk, interstitial fluid, etc.), due to the lack of intact cellular structures, only certain secretory proteins can be selected as loading control proteins.

Methods for Using Loading Control Antibodies in Western Blotting

1. For the convenient labeled internal reference: Add the HRP-labeled loading control antibody during the secondary antibody incubation step, and follow the normal experimental procedure.

2. For the conventional loading control antibody: When the molecular weight of the target protein is not significantly different from that of the selected loading control protein, first perform antibody incubation, color development, and detection for the target protein. Then, use a Strip buffer to wash off the antibodies on the membrane, and re-conduct antibody incubation, color development, and detection for the loading control protein.

3. When there is a distinct difference in molecular weight between the target protein and the selected loading control protein, you can pre-stain the membrane after membrane transfer. Based on the molecular weight of the Protein Marker, cut the membrane into two parts: one for high-molecular-weight proteins and the other for low-molecular-weight proteins, so as to separate the loading control protein from the target protein. Then, incubate the two membrane pieces with the loading control protein antibody and the target protein antibody respectively.

Felicia  Felicia is a technical support specialist at EnkiLife, with extensive professional experience in antibody development, optimization, and ELISA assay design and application. She is committed to assisting our clients in selecting suitable antibody products, optimizing ELISA experimental protocols, and resolving technical challenges encountered in the process, thereby supporting the smooth progress of their life science research projects.