How to use GAPDH antibody as a loading control

By Your Health Magazine

Loading controls are reference proteins serving as internal standards for quantifying protein levels across different experimental samples. To fulfill this role effectively, they must exhibit stable expression levels that remain unaffected by the experimental conditions. Such consistency is crucial for ensuring accurate and reliable comparisons of protein levels between samples, thus minimizing potential sources of variability and enhancing the integrity of the data.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis, playing an essential role in cellular metabolism and homeostasis. Its elevated expression levels in cells reflect its significance as a housekeeping protein, facilitating interactions with a variety of proteins, DNA, and RNA. This extensive network of interactions underlies the enzyme’s broad functional diversity. Consequently, GAPDH is frequently used as a loading control in western blotting and in numerous omics-based studies.

Why use GAPDH as a loading control?

GAPDH is widely used as a western blot loading control owing to the following key characteristics that ensure accurate and reliable protein quantification across samples.

Ubiquitous expression

GAPDH is widely produced across nearly all cell types, emphasizing its fundamental role in cellular metabolism. Its presence in diverse biological systems highlights its importance in regulating key metabolic pathways, particularly glycolysis. The enzyme’s consistent expression ensures it plays a pivotal role in maintaining cellular functions such as energy production and metabolic regulation. This broad expression makes GAPDH an ideal candidate for use as a loading control in experimental protocols, where stability across different conditions is essential. Its reliability further underscores its value in studies requiring precise normalization of protein levels.

Stable expression

GAPDH is also stably expressed across various experimental conditions. Its abundance remains relatively constant in different cell types and under diverse biological stresses, ensuring the reliability of protein quantification. Several studies confirm that GAPDH expression remains unaffected by common experimental variables, such as hypoxia, apoptosis, and cell differentiation. This consistency has led to its widespread adoption as a housekeeping gene, especially for normalizing data in protein analyses across a wide range of experimental models. However, it is important to note that in certain conditions such as cancers or stress responses, the expression of GAPDH may indeed show some variability, suggesting that careful validation is required in specialized studies.

Adaptability in different samples

GAPDH’s adaptability across various biological samples is pivotal for its role as a housekeeping gene in quantitative PCR studies. Its ability to perform consistently in different experimental setups, such as different tissue types, developmental stages, and environmental conditions, underpins its widespread use. GAPDH’s robust expression is particularly advantageous in comparative transcriptomic analyses, where sample variability could otherwise skew results. Consequently, GAPDH’s adaptability is essential for achieving accurate and reproducible gene expression data across a wide range of experimental conditions.

Protocol for using the GAPDH antibody in western blotting

Sample preparation: Tissue samples are minced and homogenized in an isolation buffer. The homogenate is first centrifuged at a low speed to remove debris, and the resulting supernatant is centrifuged at a higher speed to pellet cells. The pellet is washed, centrifuged again, and finally resuspended in a minimal volume of isolation buffer. Protein concentration is determined using the Bradford assay or bicinchoninic acid (BCA) protein assay.
SDS-PAGE and transfer: Extracted proteins are separated using 6% or 10% SDS-PAGE and transferred onto PVDF membranes. Membranes are incubated with specific primary antibodies targeting proteins of interest.
Primary incubation: Phosphorylation of GAPDH is analyzed after immunoprecipitation by incubating samples with a GAPDH antibody in a buffer containing Tris-base, NaCl, non-ionic detergent, EDTA, and protease inhibitors overnight at 4°C. Protein A/G beads are added to pull down GAPDH complexes, which are then washed, separated by SDS-PAGE, and analyzed by western blot using phospho-specific and GAPDH antibodies.
Secondary incubation: The primary incubation is followed by horse-radish peroxidase (HRP)-conjugated secondary antibodies. Phosphorylation levels are normalized to total GAPDH.
Detection: GAPDH activity can be measured using a GAPDH assay kit according to the manufacturer’s instructions. Samples are mixed with the assay master mixture in 96-well plates and absorbance is read at 690 nm by using a UV-visible plate reader. The activity is calculated as units of GAPDH per milligram of total protein based on data from at least four independent experiments.

Considerations and potential pitfalls

Although GAPDH can be used as a great loading control, certain factors can affect the reliability of its activity. These include:

Susceptibility to stress: GAPDH expression can vary under specific conditions, such as oxidative stress, hypoxia, or in certain disease states. Under low-oxygen (hypoxic) conditions, cells often increase glycolysis. The GAPDH gene promoter contains hypoxia-responsive elements and can be activated by HIF-1 (hypoxia-inducible factor). Studies have shown that in some cell types, hypoxia causes significant upregulation of GAPDH mRNA and protein. In certain endothelial and prostate cell lines, the expression of GAPDH exhibits significant oscillations in response to varying oxygen concentration. However, not all cells respond the same way. One such example is the study in glioblastoma cell lines that found no hypoxia-induced change in GAPDH expression, suggesting the regulation is cell-type specific
Variable expression: In western blotting, GAPDH and other reference proteins are commonly used to correct for uneven loading, but their reliability can be affected by physiological conditions. For example, in skeletal muscle samples from men of different ages and muscle conditions, GAPDH shows weaker linearity than β-actin and low expression levels in geriatric men. This suggests that GAPDH is not a suitable normalization control in studies involving large age differences between participants. However, GAPDH may still be reliable in studies focused on muscle wasting or differences in muscle fiber type composition.
Variability in size: Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyses have shown GAPDH peptides at multiple positions on SDS-PAGE gels, despite GAPDH’s expected molecular weight of 36 kDa. GAPDH expression is encoded by five mRNA isoforms, generating three distinct protein variants alongside a 17.6 kDa isoform originating from a noncoding RNA. These findings underscore the substantial variability in GAPDH protein expression, suggesting that it may not serve as a reliable reference gene for the normalization of protein loading in techniques such as western blotting. Consequently, caution should be exercised when using GAPDH in protein quantification studies to ensure accurate data interpretation.

Best practices for GAPDH loading

Validate stability: Using GAPDH for normalization requires accurate validation. Best practices recommend testing multiple housekeeping genes with tools to find the most stable reference.
Use appropriate controls: Consider using additional loading controls or total protein staining methods. For example, Ponceau S and Coomassie blue staining have emerged as alternatives to housekeeping genes for use as loading controls in western blots. Stain-free total protein staining offers a simpler process by eliminating staining and destaining steps.
Select high-quality antibodies: Antibody specificity is the ability of an antibody to recognize a particular epitope. Cross-reactivity occurs when an antibody binds to two similar molecules that share one or more identical epitopes. Using well-characterized antibodies with proven specificity and minimal cross-reactivity helps in removing several limitations.

Conclusion

GAPDH serves as a valuable and widely accepted loading control in western blotting and omics studies owing to its ubiquitous and generally stable expression across various tissues and experimental conditions. Its significant role in glycolysis ensures high abundance in most cells, facilitating reliable normalization of target protein expression.

However, GAPDH’s reliability can be influenced by specific physiological and pathological contexts, such as hypoxia and oxidative stress, which necessitates careful validation before usage. To achieve accurate and robust results, researchers should confirm GAPDH stability in their specific experimental setting, employ additional normalization strategies such as total protein staining methods, and select high-quality antibodies with proven specificity and minimal cross-reactivity.