Antibodies for Loading Controls

The use of housekeeping genes as loading controls

Housekeeping genes encode proteins that function to maintain basal cellular functions and ensure the survival of the cell. Due to these essential roles, housekeeping (HK) genes are understood to be stably and constitutively expressed in all cells under normal physiological conditions, regardless of developmental stage, cell cycle state, or external signal.¹ Their ubiquitous expression pattern also distinguishes them from tissue-specific genes, which can be differentially expressed in certain cell types or tissues. Evolutionary conservation of HK genes across species is also important; this helps to define genomic features and gene functions.²

While many genes can now be considered to play housekeeping roles, a small and defined subset have been historically entrenched in the literature when studying gene expression or quantifying proteins. Housekeeping genes in this context are often used as loading controls or reference genes, owing to their stable, consistent, and ubiquitous expression. They are often used to normalize for any variations in RNA or protein levels. In immunodetection applications, such as Western blotting and immunoprecipitation, antibodies to housekeeping proteins are included in experiments along with the targeted genes of interest.

List of commonly used housekeeping genes

• GAPDH — Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is among the most common housekeeping genes. It plays a central role in glycolysis by catalyzing D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate. A multifunctional protein, GAPDH is also a component of the GAIT (gamma interferon-activated inhibitor of translation) complex and exhibits nitrosylase activity.³

• Beta actin — Also known as actin beta and encoded by the ACTB gene, beta actin is a major component of actin, a highly conserved protein that polymerizes to produce cross-linked networks of filaments in the cytoplasm of cells. Beta actins are essential components of the cytoskeleton and mediators of internal cell motility.⁴

• Beta-glucuronidase — Glucuronidase beta is encoded by the GUSB gene in humans. The expressed protein is a hydrolase that degrades glycosaminoglycans, including heparan sulfate, dermatan sulfate, and chondroitin-4,6-sulfate. It is a homotetrameric protein that localizes to the lysosome.⁵

• Hsp90 — Heatshock protein 90 and related family members are highly conserved molecular chaperones that are ubiquitously expressed. They represent 1–2% of the total mammalian cellular proteins under non-stress conditions. In humans, HSP90 comprises five gene isoforms: HSP90AA1, HSP90AA2, HSP90AB1 (cytoplasmic); HSP90B1 (ER-localized); and TRAP1 (mitochondrial).⁶

• Tubulins — Alpha- and beta- tubulins form microtubules, which are a major component of the cytoskeleton. Alpha-tubulin, upon binding GTP, affects the structure of the microtubules. Beta-tubulin on the other hand regulates the dynamics of microtubules.⁷ Alpha- and beta- tubulins each consist of 9 distinct gene isoforms.

• Beta-2-microglobulin — Encoded in humans by the B2M gene, the beta-2-microglobulin protein is a component of MHC class I molecules, which are ubiquitously present on the surface of all nucleated cells of the body. Human B2M is 119 amino acids long with a mass of 13.7 kDa.⁸

• HPRT1 — Hypoxanthine phosphoribosyltransferase 1, encoded by the HPRT1 gene, is a transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. The enzyme is important in the biosynthesis of purine nucleotides.⁹

Considerations for housekeeping proteins as loading controls

Immunoblotting experiments generally involve multiple steps, such as gel loading and protein transfer, that can result in signal intensity variations of proteins. Normalization using HK proteins is a good way to help address any procedural inconsistencies. HK proteins also work well when their expression profiles are validated for the tissue or cell being investigated.¹⁰

There are certain situations, however, in which the use of typical HK proteins may not be accurate when controlling for protein expression. HK proteins levels can vary under certain conditions, such as hypoxia, serum starvation, exercise, transplantation, and cell culture density. They can also vary between different tissue types.¹⁰ Under these conditions it would be prudent to consult the literature and determine if another HK gene or protein would be better suited as a control.

More proposed housekeeping genes

More recent studies have used large RNA sequencing datasets to revisit the concept of HK genes. These aim to address the limitations of using traditional housekeeping genes, such as stable yet varying levels of expression across different tissues, and low basal expression of many genes that could be mistaken as noise.^1,11

Such studies also bring forth new candidate housekeeping genes for use as reference. For example, Eisenberg and Levanon lists a ‘set of highly uniform and strongly expressed genes’ that may be used for experimental calibration. These include C1orf43, CHMP2A, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP, and VPS29.¹

Another resource, the Housekeeping and Reference Transcript (HRT) Atlas offers an extensive database of ‘the most stable and suitable tissue selective candidate reference transcripts’ for normalization of qPCR experiments. The atlas, generated from RNA-seq data from human and mouse tissue samples, lists 2176 human and 3277 mouse HK genes.¹¹ Some notable examples incldue SSB, LAMP1, VEGFB, CD63, ATF4, GPX4, STAT3, BRD4, CD81, CDK4, HMGB1, KRAS, LAMP2, MTOR, PHF1, PTEN, SP1, and TOMM20.

References

1. Eisenberg E, Levanon EY. Human housekeeping genes, revisited [published correction appears in Trends Genet. 2014 Mar;30(3):119-20]. Trends Genet. 2013;29(10):569-574. doi:10.1016/j.tig.2013.05.010

2. Joshi CJ, Ke W, Drangowska-Way A, O'Rourke EJ, Lewis NE. What are housekeeping genes?. PLoS Comput Biol. 2022;18(7):e1010295. Published 2022 Jul 13. doi:10.1371/journal.pcbi.1010295

3. P04406 - G3P_HUMAN. UniProt. April 2024. Accessed April 2024. https://www.uniprot.org/uniprotkb/P04406/entry.

4. P60709 - ACTB_HUMAN. UniProt. April 2024. Accessed April 2024. https://www.uniprot.org/uniprotkb/P04406/entry.

5. GUSB glucuronidase beta [Homo sapiens(human)]. NCBI Gene. April 2024. Accessed April 2024. https://www.ncbi.nlm.nih.gov/gene/2990.

6. Hoter A, El-Sabban ME, Naim HY. The HSP90 Family: Structure, Regulation, Function, and Implications in Health and Disease. Int J Mol Sci. 2018;19(9):2560. Published 2018 Aug 29. doi:10.3390/ijms19092560

7. Kristensson MA. The Game of Tubulins. Cells. 2021;10(4):745. Published 2021 Mar 28. doi:10.3390/cells10040745

8. P61769 - B2MG_HUMAN. UniProt. April 2024. Accessed April 2024. https://www.uniprot.org/uniprotkb/P61769/entry.

9. HPRT1 hypoxanthine phosphoribosyltransferase 1 [Homo sapiens (human)]. NCBI Gene. April 2024. Accessed April 2024. https://www.ncbi.nlm.nih.gov/gene/3251.

10. Ghosh R, Gilda JE, Gomes AV. The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics. 2014;11(5):549-560. doi:10.1586/14789450.2014.939635

11. Hounkpe BW, Chenou F, de Lima F, De Paula EV. HRT Atlas v1.0 database: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets. Nucleic Acids Res. 2021;49(D1):D947-D955. doi:10.1093/nar/gkaa609