The DOI matches its Crossref record; 33 of the 33 references it cites were found in a registry or an index.
An old method facing a new challenge: Re-visiting housekeeping proteins as internal reference control for neuroscience research
Open accessAbstract
Study of specific target protein expression is often performed by western blotting, a commonly used method to measure the protein expression in neuroscience research by specific antibodies. Housekeeping proteins are used as an internal control for protein loading as well as reference in the western blotting analysis. This practice is based on the belief that such housekeeping genes are considered to be ubiquitously and constitutively expressed in every tissue and produce the minimal essential transcripts necessary for normal cellular function. The most commonly used housekeeping proteins are β-actin, β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). However, recent studies have shown significant variation in some housekeeping genes both at the mRNA and protein levels in various neuropathological events, such as spinal cord injury and Alzheimer's diseases. Changes of housekeeping genes are also induced by non-neuronal diseases in various tissues. Therefore, these discoveries raise a potential concern regarding whether using a housekeeping protein as an internal standard for target protein analysis is an appropriate practice. This mini review will focus on (I) the effects of neuronal and non-neuronal diseases, experimental condition, and tissues-specific roles on alteration of housekeeping genes, and (II) alternative internal standards for gene and protein expression analysis.
Overview
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Verification
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
The check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“Recent research shows that succinate is intensely produced by activated macrophages [102].”
From Protective Effects of PGC-1α Activators on Ischemic Stroke in a Rat Model of Photochemically Induced Thrombosis (2021) · cites this paper as [102]
Experimental — accuracy not yet measured
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Citations
Cites
Reversible Ponceau staining as a loading control alternative to actin in Western blots
Romero-Calvo, I., Ocón, B., Martínez-Moya, P., Suárez, M. D., Zarzuelo, A., Martínez-Augustin, O., & de Medina, F. S. (2010) · Analytical Biochemistry · doi:10.1016/j.ab.2010.02.036 · Cited by 771 · cited 1×
“While β-actin showed variability under various physiological and pathological conditions, studies using Ponceau S staining after transferring showed great correlation between Ponceau and β-actin densitometric signals in rat colon, kidney and liver samples (Romero-Calvo et al., 2010).”
Placental Stress and Pre-eclampsia: A Revised View
Redman, C. W. G., & Sargent, I. L. (2009) · Placenta · doi:10.1016/j.placenta.2008.11.021 · Cited by 611
Aldridge, G. M., Podrebarac, D. M., Greenough, W. T., & Weiler, I. J. (2008) · Journal of Neuroscience Methods · doi:10.1016/j.jneumeth.2008.05.003 · Cited by 476 · cited 2×
“The Sypro Ruby and Amido Black staining showed much better linearity with various protein loading amounts (up to 40 μg protein) than β-actin and GAPDH (Aldridge et al., 2008).”
Coomassie staining as loading control in Western blot analysis
Welinder, C., & Ekblad, L. (2011) · Journal of Proteome Research · doi:10.1021/pr1011476 · Cited by 463
Stain-Free technology as a normalization tool in Western blot analysis
Gürtler, A., Kunz, N., Gomolka, M., Hornhardt, S., Friedl, A. A., McDonald, K., Kohn, J. E., & Posch, A. (2012) · Analytical Biochemistry · doi:10.1016/j.ab.2012.10.010 · Cited by 369 · cited 1×
“One of the most recently developed technologies on detecting total protein as internal reference forwestern blotting is a staining freemethod using a pre-stained gel andmeasured by UV detection (Gürtler et al., 2013).”
Ferguson, R. E., Carroll, H. P., Harris, A. L., et al. (2005) · PROTEOMICS · doi:10.1002/pmic.200400941 · Cited by 261 · cited 5×
“…was expected to Table 1 Summary of biological event-related changes in housekeeping genes. β-actin GAPDH β-tubulin Pathological condition ↑ In kidney tumor (Ferguson et al., 2005) ↑ In spinal injury (Liu and Xu, 2006) ↑ In alcoholic hepatitis ↓ In steatosis (Boujedidi et al., 2012) ↓ In…”
Influentialβ‐Actin is not a reliable loading control in Western blot analysis
Dittmer, A., & Dittmer, J. (2006) · Electrophoresis · doi:10.1002/elps.200500785 · Cited by 174
Comparison of Stain-Free gels with traditional immunoblot loading control methodology
Colella, A. D., Chegeni, N., Tea, M. N., Gibbins, I. L., Williams, K. A., & Chataway, T. (2012) · Analytical Biochemistry · doi:10.1016/j.ab.2012.08.015 · Cited by 138 · cited 2×
“…gel under 5 different conditions such as post-transfer; post-blocking, first antibody detection, poststripping and re-blocking, and second antibody detection, showed that the quantification of protein load was linear from 10 to 40 μg total protein in all tested conditions (Colella et al., 2012).”
Housekeeping genes; expression levels may change with density of cultured cells
Greer, S. N., Honeywell, R., Geletu, M., Arulanandam, R., & Raptis, L. H. (2010) · Journal of Immunological Methods · doi:10.1016/j.jim.2010.02.006 · Cited by 128 · cited 8×
“Furthermore, those housekeeping proteins were also altered under some drug and experimental treatments and conditions (Aldridge et al., 2008; Greer et al., 2010), cell cycle phase, differentiation (Said et al.”
InfluentialMollica, J. P., Dutka, T. L., Merry, T. L., Lamboley, C. R. H., McConell, G. K., McKenna, M. J., Murphy, R. M., & Lamb, G. D. (2012) · The Journal of Physiology · doi:10.1113/jphysiol.2011.224535 · Cited by 105 · cited 3×
“…(Yu et al., 2011) Unstable in adipose tissue (Pérez-Pérez et al., 2012) ↓ In rat muscle with age (Lowe et al., 2000) Unstable in adipose tissue (Pérez-Pérez et al., 2012) ↓ With age in leukocytes (Yu et al., 2011) by interaction between VHL and hypoxia-induced factor-α gene (Ferguson et…”
Cited by
Sorted by citations
Tau protein aggregation is associated with cellular senescence in the brain
Musi, N., Valentine, J. M., Sickora, K. R., Baeuerle, E., Thompson, C. S., Shen, Q., & Orr, M. E. (2018) · Aging Cell · doi:10.1111/acel.12840 · Cited by 595
“Protein quantification was performed by normalizing to total protein concentration (Li & Shen, 2013; Moritz, 2017; Supporting Information Figure S8).”
MethodThe Design of a Quantitative Western Blot Experiment
Taylor, S. C., & Posch, A. (2014) · BioMed Research International · doi:10.1155/2014/361590 · Cited by 412
A systematic approach to quantitative Western blot analysis
Pillai‐Kastoori, L., Schutz-Geschwender, A. R., & Harford, J. A. (2020) · Analytical Biochemistry · doi:10.1016/j.ab.2020.113608 · Cited by 426
“Either type of variability may make it more difficult to reliably detect small differences and effects, leading to false-negative results [4,15,36,37].”
BackgroundAn overview of technical considerations for Western blotting applications to physiological research
Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., & Atherton, P. J. (2016) · Scandinavian Journal of Medicine and Science in Sports · doi:10.1111/sms.12702 · Cited by 433
“Other common loading controls (e.g., pan-actin and b-tubulin) have also displayed poor linear ranges in the same manner (Li & Shen, 2013).”
BackgroundLosurdo, M., Pedrazzoli, M., D’Agostino, C., Elia, C., Massenzio, F., Lonati, E., Mauri, M., Rizzi, L., Molteni, L., Bresciani, E., et al. (2020) · Stem Cells Translational Medicine · doi:10.1002/sctm.19-0327 · Cited by 272
“whose expression normalization was carried out with respect to the total amount of proteins detected by the Ponceau staining, allowing a straightforward correction for lane-to-lane variation.(38,39) For further”
Method
Metadata
- Authors
- Li, R., & Shen, Y.
- Affiliations
- Roskamp Institute (US)
- Corresponding author
- Li, Rena
- ORCID
- Venue
- Life Sciences
- Publisher
- Elsevier BV
- Published
- 2013-04
- Volume
- 92
- Issue
- 13
- Pages
- 747-751
- ISSN
- 0024-3205 (ISSN-L)
- PMC
- PMC3614345
- OpenAlex
- W2016751415
- Semantic Scholar
- fc8263e15b99660ac08ebfffcd99a11bb0edf289
- Cited by
- 186
- References
- 33
- Citations by year
- 2026: 10 · 2025: 13 · 2024: 18 · 2023: 13 · 2022: 11 · 2021: 13 · 2020: 19 · 2019: 17 · 2018: 14 · 2017: 11 · 2016: 17 · 2015: 16 · 2014: 11 · 2013: 3
- FWCI
- 1.47
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Life Sciences (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Alzheimer's Association (IIRG-07-59510, IIRG-09-61521)
- American Health Assistance Foundation (G2006-118)
- National Institute of Health (R01AG032441-01, R01AG025888-01)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Health Sciences › Health Professions › Medical Laboratory Technology › Clinical Laboratory Practices and Quality Control
- Keywords
- housekeeping proteins, Western blot, beta-actin, beta-tubulin, glyceraldehyde-3-phosphate dehydrogenase, spinal cord injury, Alzheimer's disease, neuropathology, tissue-specific gene expression
- MeSH terms
- Actins, Animals, Glyceraldehyde-3-Phosphate Dehydrogenases, Humans, Models, Molecular, Neurons, Neurosciences, Proteins, Tubulin, Genes, Essential, Gene Expression Profiling
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
