Western blotting is a very popular and sensitive protein detection system that is routinely used for multiple areas of protein research. The sensitivity of this detection system continues to improve with increasingly sensitive enhanced chemiluminescence systems and the increasing popularity of IR (infra-red) fluorophores and IR imaging systems:
Are Housekeeping Genes A Suitable Control?
The advances in IR fluorophores and IR imaging systems do not negate the use of proper controls to account for technical errors and allow for normalization and correct processing of data. Loading controls routinely rely on expression levels of “housekeeping genes” to correct for differences in protein concentration or errors in loading. Routinely used housekeeping genes include β-actin, β-tubulin or GAPDH due to their high and ubiquitous expression across cell, tissues and disease/injury types. Although popular as controls, their validity as controls has been called into question for use in:
Cultured cells:
- Greer S, Honeywell R, Geletu M, Arulanandam R, Rantis L. Housekeeping genes; expression levels may change with density of cultured cells. J Immunol Methods 2010;355:76–9
- Ferguson RE, Carroll HP, Harris A, Maher ER, Selby PJ, Banks RE. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics 2005;5:566–71
- Sikand K, Singh J, Ebron JS, Shukla GC. Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin are targets of miR-644a. PLoS One 2012;7:e47510
Tissue types:
- Ferguson RE, Carroll HP, Harris A, Maher ER, Selby PJ, Banks RE. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics 2005;5:566–71
- Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS One 2013;8:e72457
- Perez-Perez R, Lopez JA, Garcia-Santos E, Camafeita E, Gómez-Serrano M, OrtegaDelgado FJ, et al. Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity. PLoS One 2012;7:e30326.
- Moritz CP, Marz SX, Reiss R, Schulenborg T, Friauf E. Epicocconone staining a powerful loading control for Western blots. Proteomics 2014;14:162–8.
Disease/injury state:
- Ferguson RE, Carroll HP, Harris A, Maher ER, Selby PJ, Banks RE. Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies. Proteomics 2005;5:566–71
- Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS One 2013;8:e72457
- Perez-Perez R, Lopez JA, Garcia-Santos E, Camafeita E, Gómez-Serrano M, OrtegaDelgado FJ, et al. Uncovering suitable reference proteins for expression studies in human adipose tissue with relevance to obesity. PLoS One 2012;7:e30326.
- Liu NK, Xu XM. Beta-tubulin is a more suitable internal control than beta-actin in western blot analysis of spinal cord tissues after traumatic injury. J Neurotrauma 2006;23:1794–801.
- Krzystek-Korpacka M, Diakowska D, Bania J, Gamian A. Expression stability of common housekeeping genes is differently affected by bowel inflammation and cancer: implications for finding suitable normalizers for inflammatory bowel disease studies. Inflamm Bowel Dis 2014;20:1147–56.
For researchers working on secreted proteins, such as cerebral spinal fluid (CSF), the above housekeeping genes are not secreted and therefore their validity as loading controls for biological fluids can likewise be questioned (Collins et al (2015)).
Is Total Protein Staining A Suitable Alternative?
Total protein stains are used to stain membranes after protein transfer and offer the following advantages over housekeeping genes as loading controls:
- Faster: Majority of membrane stains stain within minutes, whereas blocking and antibody incubation steps for housekeeping genes can take hours,
- Affordable: Compared to the cost of primary and labeled secondary antibodies, membrane stains are very affordable. An additional cost saving is the quality of the transfer can be rapidly assessed before expensive antibodies and time are wasted on poor blots.
- Reversible: Membrane stains are reversible and do not interfere with antibody labeling and imaging.
- Save time: The use of a membrane stain allows for increased sample throughput as a flurophore channel can be used for proteins of interest as opposed to housekeeping genes.
Total Protein Staining is A Reliable Control:
- Collins, MA et al (2015) Total protein is an effective loading control for cerebrospinal fluid Western blots. J. Neurosci. Meth. 251:72
- Taylor SC, Berkelman T, Yadav G, Hammond M. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol 2013;55:217–26
- Eaton SL, Roche SL, Llavero Hurtado M, Oldknow KJ, Farquharson C, Gillingwater TH, et al. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS One 2013;8:e72457
- Moritz CP, Marz SX, Reiss R, Schulenborg T, Friauf E. Epicocconone staining a powerful loading control for Western blots. Proteomics 2014;14:162–8.
- Welinder C, Ekblad L. Coomassie staining as loading control in western blot analysis. J Proteome Res 2011;10:1416–9.
- Zeng L, Guo J, Xu HB, Huang R, Shao W, Yang L, et al. Direct Blue 71 staining as a destaining-free alternative loading control method for Western blotting. Electrophoresis 2013;34:2234–9.
- Gilda JE, Gomes AV. Stain-Free total protein staining is a superior loading control to beta-actin for Western blots. Anal Biochem 2013;440:186–8.
- Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ. The use of total protein stains as loading controls: an alternative to high-abundance single protein controls in semi-quantitative immunoblotting. J Neurosci Methods 2008;172:250–4.
- Rivero-Gutierrez B, Anzola A, Martinez-Augustin O, Sanchez de Medina F. Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting. Anal Biochem 2014;467:1–3.
- Romero-Calvo I, Ocon B, Martinez-Moya P, Suarez MD, Zarzuelo A, MartínezAugustin O, et al. Reversible ponceau staining as a loading control alternative to actin in western blots. Anal Biochem 2010;401:318–20.
- Klein D, Kern RM, Sokol RZ. A method for quantification and correction of proteins after transfer to immobilization membranes. Biochem Mol Biol Int 1995;36:59–66.
So it appears that total protein stains offer significant advantages over housekeeping genes as loading controls for Western blotting.
One major question still remains and has arisen due to the new IR imaging systems:
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