Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an enzyme involved in glycolysis, the process cells use to convert sugar into energy. Because this function is fundamental to life, the GAPDH protein is found in nearly all cell types and tissues. This constant presence has given the enzyme a second, unrelated role in scientific laboratories: a standard for comparison in experiments. For decades, its seemingly unwavering expression has made it a go-to benchmark for researchers measuring changes in other molecules. This article explores how and why GAPDH is used as a research control and examines the growing awareness of its limitations.
The Role of a Housekeeping Gene
In any experiment, a control is a standard used to minimize the effects of variables other than the one being tested. In molecular biology, this is often a “loading control.” Imagine comparing two cake recipes to see which is sweeter; if you use different amounts of sugar for each, your test is flawed. A loading control ensures that the starting amount of material in each sample is the same.
Housekeeping genes are thought to be expressed at a consistent level in virtually all cells under any condition because their functions are necessary for basic cellular survival. Scientists leverage this presumed stability to normalize their data. By measuring the level of a housekeeping gene product, like the GAPDH protein, they can confirm that an equal amount of total sample was loaded for each condition.
If the housekeeping gene level is consistent across all samples, researchers can be more confident that any differences they see in their protein or gene of interest are real biological changes. These changes would not be artifacts of pipetting errors or inconsistencies in sample preparation. For many years, GAPDH has been one of the most widely used housekeeping genes for this purpose.
GAPDH in Common Laboratory Techniques
GAPDH is a common control in two foundational laboratory techniques: Western blotting for analyzing proteins, and quantitative polymerase chain reaction (qPCR) for analyzing gene expression. Both methods rely on the assumption that GAPDH levels remain constant to ensure the accuracy of the results.
In Western blotting, researchers separate proteins from a mixture based on their size. After transferring them to a membrane, they use specific antibodies to detect their protein of interest. To ensure an observed difference is a true biological effect, they also probe the membrane with an antibody for a housekeeping protein. The amount of GAPDH detected serves as a baseline, confirming an equal quantity of total protein was loaded in each sample.
Similarly, qPCR is a technique used to measure the amount of messenger RNA (mRNA) from a specific gene, which indicates its activity. The expression of a gene of interest is measured relative to the expression of a housekeeping gene assumed to be stable, with GAPDH being a frequent choice. By comparing the target gene’s mRNA to that of GAPDH mRNA, researchers can determine if a treatment caused the gene to become more or less active.
Limitations and Considerations
Despite its history as a reliable standard, evidence shows that GAPDH is not as stable as once believed. Its expression can change significantly under various experimental conditions, complicating its use as a control. This variability means the scientific “ruler” being used to measure changes might itself be changing, leading to inaccurate conclusions.
A primary factor that alters GAPDH expression is hypoxia, or low oxygen levels. Under hypoxic conditions, common in solid tumors, GAPDH expression can increase as the cell shifts its metabolism. Using GAPDH as a control in cancer studies involving hypoxia could mask a true increase or create a false decrease in the target protein’s level.
Baseline GAPDH levels can also differ dramatically between cell types, such as muscle and brain tissue, due to varying metabolic demands. Disease states, including metabolic disorders and cancers, can directly alter its expression. Even drug treatments being tested can affect GAPDH levels, compromising its function as a neutral benchmark and requiring careful validation of controls.
Alternative Housekeeping Controls
The discovery that GAPDH expression can be unstable has prompted researchers to be more meticulous in selecting controls. The best practice is to validate the stability of a chosen control for the specific cell type and experimental conditions being studied. This involves testing several potential housekeeping genes to find one that shows the least variability.
Several other proteins and genes are now used as alternatives or supplements to GAPDH. Beta-actin (ACTB) and Tubulin (TUB) are two popular choices; both are structural proteins involved in the cell’s cytoskeleton. These proteins are often, but not always, more stable than the metabolically-involved GAPDH.
In gene expression studies using qPCR, 18S ribosomal RNA (rRNA) is another alternative. As a component of the ribosome, 18S rRNA is abundant and often stably expressed. A more rigorous approach is to use the geometric mean of multiple housekeeping genes for normalization. This method averages out the slight variations in any single control, providing a more reliable baseline.