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.
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
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?
Recent research shows that succinate is intensely produced by activated macrophages.
“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]
The sections on housekeeping proteins and pathological changes discuss β-actin, β-tubulin, and GAPDH, not succinate production. Searches for succinate and activated macrophages found no passage in our copy addressing the claim; the paper therefore cannot verify it. (LLM, conf=0.77)
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.
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× · see citing works
“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 601 · see citing works
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× · see citing works
“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 · see citing works
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× · see citing works
“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× · see citing works
“…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 168 · see citing works
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× · see citing works
“…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× · see citing works
“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× · see citing works
“…(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…”
Sikand, K., Singh, J., Ebron, J. S., & Shukla, G. C. (2012) · PLoS ONE · doi:10.1371/journal.pone.0047510 · Cited by 97 · cited 4× · see citing works
“Recent studies also reported that microRNA inhibition or over- expression in vitro might also regulate β-actin and GAPDH expression (Sikand et al., 2012), suggesting β-actin and GAPDH should not be used as an internal control for normalization of microRNA targeted mRNA expression.”
InfluentialLu, S., Gu, X., Hoestje, S. M., & Epner, D. E. (2002) · Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression · doi:10.1016/s0167-4781(01)00359-1 · Cited by 94 · cited 3× · see citing works
“by interaction between VHL and hypoxia-induced factor-α gene (Ferguson et al., 2005; Lu et al., 2002).”
Dang, W., & Sun, L. (2011) · Fish & Shellfish Immunology · doi:10.1016/j.fsi.2010.12.028 · Cited by 91 · cited 2× · see citing works
“They detected these housekeeping genes in the liver, spleen, kidney, gill, heart and brain and reported that RPSD is the most stable gene across tissue types under normal physiological conditions and during bacterial infection, while β-actin might be used as an internal control for the normalization of gene expression…”
Stability of reference proteins in human placenta: General protein stains are the benchmark
Lanoix, D., St‐Pierre, J., Lacasse, A., Viau, M., Lafond, J., & Vaillancourt, C. (2012) · Placenta · doi:10.1016/j.placenta.2011.12.008 · Cited by 74 · see citing works
Liu, N., & Xu, X. (2006) · Journal of Neurotrauma · doi:10.1089/neu.2006.23.1794 · Cited by 71 · cited 3× · see citing works
“Studies showed that spinal cord injury induced more than a 2 fold increase in β-actin expression while no statistically significant difference was found in β-tubulin expression after the injury compared with sham-operated controls (Liu and Xu, 2006).”
Glyceraldehyde-3-Phosphate Dehydrogenase Varies With Age in Glycolytic Muscles of Rats
Lowe, D. A., Degens, H., Chen, K. D., et al. (2000) · The Journals of Gerontology Series A · doi:10.1093/gerona/55.3.b160 · Cited by 65 · cited 5× · see citing works
“, 2009) or proliferation status, and age (Lowe et al., 2000).”
InfluentialExpression of four housekeeping proteins in elderly patients with schizophrenia
Bauer, D., Haroutunian, V. H., McCullumsmith, R. E., & Meador‐Woodruff, J. H. (2009) · Journal of Neural Transmission · doi:10.1007/s00702-008-0143-3 · Cited by 61 · cited 2× · see citing works
“…studies of schizophrenia as there were no differences in the expression of those housekeeping proteins between schizophrenia and comparison groups (Bauer et al., 2009), although a recent study demonstrated that β-tubulin protein levels were decreased in the anterior cingulated cortex, increased…”
Detection of Proteins on Blot Membranes
Harper, S. L., & Speicher, D. W. (1995) · Current Protocols in Protein Science · doi:10.1002/0471140864.ps1008s00 · Cited by 57 · see citing works
Quantification of Gene Expression after Painful Nerve Injury: Validation of Optimal Reference Genes
Bangaru, M. L. Y., Park, F., Hudmon, A., et al. (2011) · Journal of Molecular Neuroscience · doi:10.1007/s12031-011-9628-x · Cited by 47 · cited 1× · see citing works
“…gene expression after nerve injury was also reported by other independent research groups, that compared β-actin and β-tubulin, GAPDH as the one most stable housekeeping genes for normalizing gene expression for qRT-PCR analysis in the context of peripheral nerve injury (Bangaru et al., 2012).”
Lack of expression and function of erythropoietin receptors in the kidney
Elliott, S., Busse, L. A., Swift, S. E., McCaffery, I., Rossi, J. M., Kassner, P. D., & Begley, C. G. (2011) · Nephrology Dialysis Transplantation · doi:10.1093/ndt/gfr698 · Cited by 40 · cited 1× · see citing works
“The staining free gel method has been used as loading control in several recent publications (Mollica et al., 2012; Larkins et al., 2012; Elliott et al., 2012).”
Absence of GAPDH regulation in tumor-cells of different origin under hypoxic conditions in – vitro
Said, H. M., Polat, B., Hagemann, C., Anacker, J., Flentje, M. P., & Vordermark, D. (2009) · BMC Research Notes · doi:10.1186/1756-0500-2-8 · Cited by 40 · cited 4× · see citing works
“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., 2009) or proliferation status, and age (Lowe et al., 2000).”
InfluentialHousekeeping Gene Variability in the Liver of Alcoholic Patients
Boujedidi, H., Bouchet‐Delbos, L., Cassard, A.-M. A. D. T., et al. (2011) · Alcoholism Clinical and Experimental Research · doi:10.1111/j.1530-0277.2011.01627.x · Cited by 38 · see citing works
Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue
Gebhardt, F. M., Scott, H. A., & Dodd, P. R. (2010) · Alzheimer s & Dementia · doi:10.1016/j.jalz.2009.11.002 · Cited by 36 · cited 6× · see citing works
“…kidney tumor (Ferguson et al., 2005) ↑ In spinal injury (Liu and Xu, 2006) ↑ In alcoholic hepatitis ↓ In steatosis (Boujedidi et al., 2012) ↓ In Alzheimer's brain (Gebhardt et al., 2010) ↑ In alcoholic hepatitis ↓ In steatosis (Boujedidi et al., 2012) ↓ In Alzheimer's brain (Gebhardt et al.,…”
InfluentialLarkins, N. T., Murphy, R. M., & Lamb, G. D. (2012) · American Journal of Physiology-Cell Physiology · doi:10.1152/ajpcell.00180.2012 · Cited by 31 · cited 1× · see citing works
“The staining free gel method has been used as loading control in several recent publications (Mollica et al., 2012; Larkins et al., 2012; Elliott et al., 2012).”
Pérez‐Pérez, R., López, J. A., García‐Santos, E., Camafeita, E., Gómez‐Serrano, M., Ortega, F. J., Ricart, W., Fernández‐Real, J. M., & Peral, B. (2012) · PLoS ONE · doi:10.1371/journal.pone.0030326 · Cited by 28 · cited 6× · see citing works
“, 2011) Unstable in adipose tissue (Pérez-Pérez et al., 2012)”
InfluentialUse of Sample Mixtures for Standard Curve Creation in Quantitative Western Blots
Suzuki, O., Koura, M., Noguchi, Y., et al. (2011) · EXPERIMENTAL ANIMALS · doi:10.1538/expanim.60.193 · Cited by 28 · cited 2× · see citing works
“Given that housekeeping protein bands often appear very thick and dark in intensity on many published papers, it is possible that the chemiluminescence signal of the housekeeping protein can be easily saturated in this range and loses the credibility for quantization (Colella et al., 2012; Suzuki et al., 2011).”
Regional differences in expression of β-tubulin isoforms in schizophrenia
Moehle, M. S., Ludueña, R. F., Haroutunian, V. H., Meador‐Woodruff, J. H., & McCullumsmith, R. E. (2012) · Schizophrenia Research · doi:10.1016/j.schres.2011.12.010 · Cited by 25 · cited 5× · see citing works
“…a recent study demonstrated that β-tubulin protein levels were decreased in the anterior cingulated cortex, increased in the dorsolateral prefrontal cortex and saw no change in the hippocampus in schizophrenia (Moehle et al., 2012), suggesting a disease-associated major cytoskeleton protein.”
InfluentialYu, H., Kuo, H., Huang, H., et al. (2011) · Analytical Biochemistry · doi:10.1016/j.ab.2011.01.037 · Cited by 25 · cited 4× · see citing works
“Tissue-specific condition ↓ With age in leukocytes (Yu et al., 2011) Unstable in adipose tissue (Pérez-Pérez et al.”
InfluentialEvaluation of reference genes for quantitative PCR analysis of mouse lymphocytes
Albershardt, T. C., Iritani, B. M., & Ruddell, A. (2012) · Journal of Immunological Methods · doi:10.1016/j.jim.2012.07.020 · Cited by 16 · cited 3× · see citing works
“12 μg of total loading protein (Dittmer and Dittmer, 2006) ↓ In VHL transfection (Ferguson et al., 2005; Lu et al., 2002) ↓ In miRNA treatment (Sikand et al., 2012) Extensive variables in qPCR (Albershardt et al., 2012) ↑ With cell confluence (10–100”
Application of SYPRO Ruby- and Flamingo-stained polyacrylamide gels to Western blot analysis
Hagiwara, M., Kobayashi, K., Tadokoro, T., et al. (2009) · Analytical Biochemistry · doi:10.1016/j.ab.2009.03.037 · Cited by 14 · see citing works
Hagiwara, M., Kobayashi, K., Tadokoro, T., et al. (2009) · Analytical Biochemistry · doi:10.1016/j.ab.2009.10.032 · Cited by 12 · cited 1× · see citing works
“…be used for target protein blotting, recently modified Sypro Ruby- and Flamingo-staining methods provide an application of completed western blotting after gel staining (Hagiwara et al., 2010), suggesting a potential usage of Sypro Ruby and Amido Black for protein loading control in western blot.”
A fluorescence-based Coomassie Blue protocol for two-dimensional gel-based proteomics
Yang, Y., Wang, J., Bu, D., et al. (2010) · Biotechnology Letters · doi:10.1007/s10529-010-0404-8 · Cited by 7 · cited 2× · see citing works
“The sensitivity of newly developed “visible Sypro” staining is compatible with mass spectrometry (Yang et al., 2011) such as detection protein range improving up to 2.5 ng on SDS-PAGE gel.”
InfluentialRen, G., Okerberg, C. K., & Mathews, S. T. (2012) · Methods in molecular biology · doi:10.1007/978-1-61779-821-4_57 · Cited by 6 · cited 1× · see citing works
“Studies showed that both staining have detection sensitivity around 50 ng/protein band (Harper and Speicher, 2001; Ren et al., 2012).”
Flagged first, then by citations
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 · see citing works
“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 · see citing works
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 407 · see citing works
“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 407 · see citing works
“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 265 · see citing works
“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”
MethodBrouwer, K. L. R., Aleksunes, L. M., Brandys, B., Giacoia, G. P., Knipp, G. T., Lukacova, V., Meibohm, B., Nigám, S. K., Rieder, M. J., de Wildt, S. N., et al. (2015) · Clinical Pharmacology & Therapeutics · doi:10.1002/cpt.176 · Cited by 185 · see citing works
“Another emerging issue is that mRNA expression of housekeeper genes can vary significantly, thus calling into question quantitative data from qRT-PCR studies normalized to one control.(56,57) These concerns may be alleviated by the use of RNA-sequencing to quantify human transporter ontogeny, as has been performed in…”
BackgroundVigelsø, A., Dybboe, R., HANSEN, C. N., Dela, F., Helge, J. W., & Guadalupe‐Grau, A. (2014) · Journal of Applied Physiology · doi:10.1152/japplphysiol.00840.2014 · Cited by 117 · see citing works
“, fiber type distribution (5, 13), aging (12), and muscle wasting by immobilization (16)].”
InfluentialTotal protein or high-abundance protein: Which offers the best loading control for Western blotting?
Thacker, J. S., Yeung, D. H., Staines, R. W., et al. (2015) · Analytical Biochemistry · doi:10.1016/j.ab.2015.11.022 · Cited by 114 · see citing works
“…used “housekeeping” proteins can fluctuate in response to various stimuli, different stages of cellular growth and differentiation, and certain disease states (see reviews by Ruan and Lai, 2007 and Li and Shen, 2013; also, Calvo et al., 2008 and Ferguson M AN US CR IP T AC CE PT ED 4 et al., 2005).”
BackgroundMelón, L. C., Hooper, A., Yang, X., Moss, S. J., & Maguire, J. (2018) · Psychoneuroendocrinology · doi:10.1016/j.psyneuen.2017.12.003 · Cited by 106 · see citing works
“Optical density measurements were performed using NIH ImageJ software and normalized to total protein levels (25μg total protein) rather than a housekeeping protein, which have shown variability in expression levels (Li and Shen 2013).”
MethodAlessio, E., Buson, L., Chemello, F., Peggion, C., Grespi, F., Martini, P. G. V., Massimino, M. L., Pacchioni, B., Millino, C., Romualdi, C., et al. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz007 · Cited by 82 · see citing works
Chemello, F., Grespi, F., Zulian, A., Cancellara, P., Hébert-Chatelain, É., Martini, P. G. V., Bean, C., Alessio, E., Buson, L., Bazzega, M., et al. (2019) · Cell Reports · doi:10.1016/j.celrep.2019.02.105 · Cited by 72 · see citing works
Sharafeldin, M., Chen, T., Özkaya, G. U., Choudhary, D., Molinolo, A., Gutkind, J. S., & Rusling, J. F. (2020) · Biosensors and Bioelectronics · doi:10.1016/j.bios.2020.112681 · Cited by 69 · see citing works
“The ability to detect sub-fg levels of DSG3, VEGF-A and VEGF-C promised single cell analyses. β-Tub, a loading control in western blots (Liu and Xu, 2006; Li and Shen, 2013), was measured as a cell counting marker for highly diluted samples.”
BackgroundMuelas, M. W., Mughal, F., O’Hagan, S., Day, P. J., & Kell, D. B. (2019) · Scientific Reports · doi:10.1038/s41598-019-54288-7 · Cited by 65 · see citing works
Xing, G., Barry, E. S., Benford, B., Grunberg, N. E., Li, H., Watson, W. D., & Sharma, P. (2013) · Frontiers in Neurology · doi:10.3389/fneur.2013.00196 · Cited by 66 · see citing works
“Due to the lack of an appropriate housekeeping mitochondrial protein (33, 34), WB band intensity was expressed as fold change relative to naives, but was not normalized to an internal control.”
BackgroundComparative Proteomics Analysis of Human Liver Microsomes and S9 Fractions
Wang, X., He, B., Shi, J., Li, Q., & Zhu, H. (2019) · Drug Metabolism and Disposition · doi:10.1124/dmd.119.089235 · Cited by 64 · see citing works
“, 2011), which could lead to inaccurate protein expression measurements (Sullivan-Gunn et al., 2011; Li and Shen, 2013).”
BackgroundAlinezhad, S., Väänänen, R., Mattsson, J., Li, Y., Tallgrén, T., Tong-Ochoa, N., Bjartell, A. S., Åkerfelt, M., Taimen, P., Boström, P. J., et al. (2016) · PLoS ONE · doi:10.1371/journal.pone.0155901 · Cited by 65 · see citing works
“However, the reliability of housekeeping genes for normalization has often been questioned due to their instability during storage and inconsistent expression changes in different disease conditions [22,23].”
BackgroundMelón, L., Hammond, R., Lewis, M., & Maguire, J. (2018) · Frontiers in Endocrinology · doi:10.3389/fendo.2018.00703 · Cited by 62 · see citing works
“Optical density measurements were performed using NIH ImageJ software and normalized to total protein levels (25 μg total protein) rather than a housekeeping protein, which have shown variability in expression levels (31).”
MethodActive Tensile Modulus of an Epithelial Monolayer
Vincent, R. R. R., Bazellières, E., Gonzalez, C. P., et al. (2015) · Physical Review Letters · doi:10.1103/physrevlett.115.248103 · Cited by 63 · see citing works
Hephaestin and Ceruloplasmin Play Distinct but Interrelated Roles in Iron Homeostasis in Mouse Brain
Jiang, R., Hua, C., Wan, Y., Jiang, B., Hu, H., Zheng, J., Fuqua, B. K., Dunaief, J. L., Anderson, G. J., David, S., et al. (2015) · Journal of Nutrition · doi:10.3945/jn.114.207316 · Cited by 60 · see citing works
“Expression levels were normalized to that of the housekeeping genes GAPDH or hypoxanthine phosphoribosyltransferase (HPRT) (32, 33).”
MethodSiniscalco, D., Bradstreet, J. J., Cirillo, A., & Antonucci, N. (2014) · Journal of Neuroinflammation · doi:10.1186/1742-2094-11-78 · Cited by 58 · see citing works
“To assess equal loading, protein levels were normalized with respect to the signal obtained with Coomassie Blue staining, as previously reported [31].”
MethodAdamski, M. G., Gumann, P., & Baird, A. E. (2014) · PLoS ONE · doi:10.1371/journal.pone.0103917 · Cited by 56 · see citing works
“Therefore, reference gene selection requires additional experiments to validate gene expression stability under different experimental conditions [6–12,14].”
BackgroundThe impact of age and frailty on ventricular structure and function in C57BL/6J mice
Feridooni, H. A., Kane, A. E., Ayaz, O., Boroumandi, A., Polidovitch, N., Tsushima, R. G., Rose, R. A., & Howlett, S. E. (2017) · The Journal of Physiology · doi:10.1113/jp274134 · Cited by 56 · see citing works
“Ponceau S was used as the loading control as there is evidence to suggest that standard housekeeping proteins, such as GAPDH and β-actin, are affected by age (Lowe et al. 2000; Li & Shen, 2013; Mennes et al. 2014).”
BackgroundMethodMilani, C., Farina, F., Botto, L., Massimino, L., Lonati, E., Donzelli, E., Ballarini, E., Crippa, L., Marmiroli, P., Bulbarelli, A., et al. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21103699 · Cited by 50 · see citing works
“Recent studies reported that total protein staining represents the actual loading amount more accurately than a housekeeping protein due to minor procedural and biological variations [89,90].”
BackgroundAdaptive structural changes in the motor cortex and white matter in Parkinson’s disease
Fu, Y., Zhou, L., Li, H., Hsiao, J.-H. T., Li, B., Tanglay, O., Auwyang, A. D., Wang, E., Feng, J., Kim, W. S., et al. (2022) · Acta Neuropathologica · doi:10.1007/s00401-022-02488-3 · Cited by 51 · see citing works
Walker, J. M., Kazempour Dehkordi, S., Fracassi, A., Vanschoiack, A., Pavenko, A., Taglialatela, G., Woltjer, R., Richardson, T. E., Zare, H., & Orr, M. E. (2022) · Acta Neuropathologica Communications · doi:10.1186/s40478-022-01324-9 · Cited by 59 · see citing works
“Digital counts were first normalized with internal controls (GAPDH, Histone H3, and S6) for system variation, although these have been shown to have altered expression in neurofibrillary tangles in Alzheimer disease [25, 27], therefore, samples were normalized to spiked-in External RNA Controls Consortium (ERCC) RNA c…”
MethodAithal, M. G. S., & Rajeswari, N. (2015) · Brain Tumor Research and Treatment · doi:10.14791/btrt.2015.3.1.24 · Cited by 47 · see citing works
“Several publications have shown that Beta-actin and GAPDH vary considerably and are thus unsuitable references for RNA transcription analysis [9-12].”
BackgroundWalker, J. M., Orr, M. E., Orr, T. C., Thorn, E. L., Christie, T. D., Yokoda, R. T., Vij, M., Ehrenberg, A. J., Marx, G. A., McKenzie, A. T., et al. (2023) · Alzheimer s & Dementia · doi:10.1002/alz.13484 · Cited by 47 · see citing works
“28,29 ROIs were manually re-inspected for accuracy after nCounter analysis using both the morphology and quantitative markers; ROIs containing NFTs were checked to ensure higher levels of p-tau compared to ROIs containing non-NFT-bearing neurons.”
MethodDownregulation of protein phosphatase 2A by apolipoprotein E: Implications for Alzheimer's disease
Theendakara, V. P., Bredesen, D. E., & Rao, R. V. (2017) · Molecular and Cellular Neuroscience · doi:10.1016/j.mcn.2017.07.002 · Cited by 46 · see citing works
“Tissue specific alterations of housekeeping genes like β-actin, GAPDH and β-tubulin especially in pathological conditions have been reported that have caused problems in data acquisition (Li and Shen, 2013).”
BackgroundMartins, I., Carvalho, P., de Vries, M. G., Teixeira-Pinto, A. M., Wilson, S. P., Westerink, B. H. C., & Tavares, I. (2015) · Anesthesiology · doi:10.1097/aln.0000000000000749 · Cited by 44 · see citing works
Jackson, T. C., Du, L., Janesko‐Feldman, K. L., et al. (2015) · Journal of Cerebral Blood Flow & Metabolism · doi:10.1038/jcbfm.2014.242 · Cited by 40 · see citing works
Li, S.-W., Yu, H., Sheen, J., Tiao, M., Tain, Y., Lin, I.-C., Lin, Y., Chang, K., Tsai, C., & Huang, L. (2017) · Oncotarget · doi:10.18632/oncotarget.22960 · Cited by 40 · see citing works
“The IOD was baseline adjusted from Ponceau S-staining to correct any variations in total protein loading and an internal standard [76, 80, 81].”
MethodLemmens, K., Bollaerts, I., Bhumika, S., Groef, L. D., houcke, J. V., Darras, V., Hove, I. V., & Moons, L. (2015) · The Journal of Comparative Neurology · doi:10.1002/cne.23920 · Cited by 38 · see citing works
“bands were normalized to corresponding bands of the total protein stain to correct for interlane variability of protein loading (Li and Shen, 2013).”
MethodHu, X., Du, S., Yu, J., et al. (2016) · Oncotarget · doi:10.18632/oncotarget.11439 · Cited by 38 · see citing works
“Varied methods of staining are available when detecting the total protein, such as SYPRO Ruby staining, Coomassie Brilliant Blue staining, Reversible Ponceau S staining or using staining-free gels instead [27].”
MethodMetaproteomic portrait of the healthy human gut microbiota
Tanca, A., Palomba, A., Fiorito, G., Abbondio, M., Pagnozzi, D., & Uzzau, S. (2024) · npj Biofilms and Microbiomes · doi:10.1038/s41522-024-00526-4 · Cited by 38 · see citing works
“GAPDH is a moonlighting proteinthatfunctionsasglycolyticenzymeaswellasuracilDNAglycosylaseandhasbeenusedfordecadesasahousekeepingproteininproteomicstudiesoneukaryoticcellandtissues ,althoughitsuseasinternalstandard shouldbeexaminedcarefullyinrelationtotheexperimentalconditionsand diseasestate 29 .”
Walter, R. F. H., Werner, R., Vollbrecht, C., et al. (2016) · PLoS ONE · doi:10.1371/journal.pone.0165181 · Cited by 36 · see citing works
Agarwal, G., Roy, A., Kumar, H., & Srivastava, A. (2022) · Biomaterials Advances · doi:10.1016/j.bioadv.2022.212971 · Cited by 37 · see citing works
Crivellaro, G., Tottene, A., Vitale, M., Melone, M., Casari, G., Conti, F., Santello, M., & Pietrobon, D. (2021) · Neurobiology of Disease · doi:10.1016/j.nbd.2021.105419 · Cited by 35 · see citing works
“To control protein loading, β-actin was used as housekeeping protein (Li and Shen, 2013; Capuani et al., 2016).”
Lee, H., Jo, J., Hong, H.-H., Kim, K. K., Park, J., Cho, S., & Park, C. (2016) · PROTEOMICS · doi:10.1002/pmic.201500344 · Cited by 34 · see citing works
Walter, R. F. H., Mairinger, F. D., Wohlschlaeger, J., et al. (2013) · Pathology - Research and Practice · doi:10.1016/j.prp.2013.09.007 · Cited by 34 · see citing works
Sex-dependent effects of developmental hypoxia on cardiac mitochondria from adult murine offspring
Hellgren, K. T., Premanandhan, H., Quinn, C. J., Trafford, A. W., & Galli, G. L. J. (2020) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2020.11.004 · Cited by 34 · see citing works
Fuller, H. R., Gillingwater, T. H., & Wishart, T. M. (2016) · Neuromuscular Disorders · doi:10.1016/j.nmd.2016.06.004 · Cited by 33 · see citing works
“Documented variation in the mRNA and protein levels of GAPDH in other neuropathological events have been reported [33], and suggest that it may play a key role in neurodegenerative processes.”
A neurogenic perspective of Sarcopenia: time course study of sciatic nerves from aging mice
Krishnan, V. S., White, Z., McMahon, C. D., Hodgetts, S. I., Fitzgerald, M., Shavlakadze, T., Harvey, A. R., & Grounds, M. D. (2016) · Journal of Neuropathology & Experimental Neurology · doi:10.1093/jnen/nlw019 · Cited by 32 · see citing works
Challenges of MS‐based small extracellular vesicles proteomics
Fochtman, D., Marczak, Ł., Pietrowska, M., & Wojakowska, A. (2024) · Journal of Extracellular Vesicles · doi:10.1002/jev2.70020 · Cited by 33 · see citing works
Khatib, T., Chisholm, D. R., Whiting, A., Platt, B., & McCaffery, P. J. A. (2019) · Journal of Alzheimer s Disease · doi:10.3233/jad-190931 · Cited by 28 · see citing works
“The levels of our proteins of interest were normalized to total protein levels rather than using a specific reference protein such as -actin [45–49], because the housekeeping protein levels may vary depending on disease state especially in neuropathological events [50].”
MethodWei, Y., Zhang, J., Xia, Y., Peng, X., Xu, S., Chang, H., Wang, H., & Gao, Y. (2019) · Frontiers in Physiology · doi:10.3389/fphys.2019.01449 · Cited by 30 · see citing works
Tian, X., Zhu, H., Du, S., et al. (2020) · Journal of Pain · doi:10.1016/j.jpain.2020.03.009 · Cited by 29 · see citing works
“After protein concentration was measured, equal amounts of total protein from each sample were loaded.(22) The samples were heated at 99°C for 5 minutes and loaded onto a 4 to 15% stacking/7.”
MethodZhang, J., Jia, G., Xue, P., et al. (2021) · Therapeutic Advances in Chronic Disease · doi:10.1177/2040622321995685 · Cited by 28 · see citing works
Saddala, M. S., Lennikov, A., Grab, D. J., Liu, G., Tang, S., & Hu, H. (2018) · Scientific Reports · doi:10.1038/s41598-018-34955-x · Cited by 27 · see citing works
Hsu, M., Sheen, J., Lin, I.-C., Yu, H., Tiao, M., Tain, Y., & Huang, L. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21103428 · Cited by 26 · see citing works
“The IOD was baseline that adjusted from Ponceau S-staining to correct possibly variations in total protein loading [66,69,70].”
MethodWang, Z., Xu, J.-H., Mou, J., Kong, X.-T., Zou, J.-W., Xue, H., Wu, M., & Lai-xiang, X. (2020) · Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology · doi:10.1016/j.cbpa.2020.110766 · Cited by 24 · see citing works
Cartagena, C. M., Mountney, A., Hwang, H., Swiercz, A. P., Rammelkamp, Z., Boutté, A., Shear, D. A., Tortella, F., & Schmid, K. E. (2016) · PLoS ONE · doi:10.1371/journal.pone.0158576 · Cited by 23 · see citing works
“β-actin levels were measured for each blot but not used for normalization purposes due to significant increases at later time points (3d, 7d) post-injury (S5 Fig) as has been shown in other neuropathology models [32].”
MethodZhang, J., Li, X., Ismail, F., Xu, S., Wang, Z., Peng, X., Yang, C., Chang, H., Wang, H., & Gao, Y. (2019) · Cells · doi:10.3390/cells9010042 · Cited by 24 · see citing works
“As described previously [32,33], the original images captured with no gain were stored.”
A Proteomic Study of Memory After Imprinting in the Domestic Chick
Meparishvili, M., Nozadze, M., Margvelani, G., McCabe, B. J., & Solomonia, R. O. (2015) · Frontiers in Behavioral Neuroscience · doi:10.3389/fnbeh.2015.00319 · Cited by 23 · see citing works
“…to actin or any other housekeeping protein because it cannot be guaranteed that such proteins are unaffected by imprinting (see also Dittmer and Dittmer, 2006; Li and Shen, 2013; Ghosh et al., 2014; Chen and Xu, 2015 for discussion of the unreliability of normalization to housekeeping proteins).”
BackgroundMethodSnow, W. M., Pahlavan, P. S., Djordjevic, J., McAllister, D., Platt, E. E., Alashmali, S. M., Bernstein, M. J., Suh, M., & Albensi, B. C. (2015) · Frontiers in Molecular Neuroscience · doi:10.3389/fnmol.2015.00070 · Cited by 23 · see citing works
“…control for normalization of Western blot data due to studies documenting experimentally-induced changes in actin levels (Gilda and Gomes, 2013; Li and Shen, 2013; RiveroGutierrez et al., 2014), we investigated putative differences in actin as a function ofMWM-training in the mouse hippocampus.”
BackgroundResultMcInerney, M. P., Pan, Y., Short, J. L., & Nicolazzo, J. A. (2017) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2016.12.017 · Cited by 22 · see citing works
“It has been w ell reported in the literature that house32 keeping protein expression levels fluctuate more th an was previously understood, and that 33 their high expression levels lead to saturated sign als.(23-26) Since each cell can possess only one 34”
BackgroundChin, E. R., Chen, D., Bobyk, K. D., & Mázala, D. A. G. (2014) · American Journal of Physiology-Cell Physiology · doi:10.1152/ajpcell.00237.2013 · Cited by 22 · see citing works
Xu, L., Luo, H., Wang, R., et al. (2019) · BMC Medical Genetics · doi:10.1186/s12881-019-0867-y · Cited by 22 · see citing works
López-Toledo, G., Silva‐Lucero, M., Herrera‐Díaz, J., García, D.-E., Arias‐Montaño, J., & Cárdenas‐Aguayo, M. D. C. (2022) · Frontiers in Aging Neuroscience · doi:10.3389/fnagi.2022.921573 · Cited by 19 · see citing works
Soares, D. D. S., Pinto, G. H., Lopes, A. G., Caetano, D. S. L., Nascimento, T. G., Andrades, M. É., Clausell, N., Rohde, L. E. P., Leitão, S. A. T., & Biolo, A. (2019) · American Journal of Physiology-Regulatory, Integrative and Comparative Physiology · doi:10.1152/ajpregu.00205.2018 · Cited by 21 · see citing works
“Results are expressed as percentage of change from baseline in arbitrary units (%AU) using the receptor densitometry-to-Coomassie blue densitometry ratio (13, 24, 28).”
MethodKrekeler, B. N., Weycker, J. M., & Connor, N. P. (2020) · Dysphagia · doi:10.1007/s00455-020-10105-2 · Cited by 19 · see citing works
López-Landavery, E. A., Portillo-López, A., Gallardo‐Escárate, C., & del Río-Portilla, M. Á. (2014) · Gene · doi:10.1016/j.gene.2014.08.002 · Cited by 18 · see citing works
Sutton, W. J. H., Branham, P. J., Williamson, Y. M., Cooper, H. C., Najjar, F. N., Pierce, C. L., Barr, J. R., & Williams, T. L. (2023) · Vaccine · doi:10.1016/j.vaccine.2023.04.044 · Cited by 18 · see citing works
“Protein expression is typically estimated by comparison against the levels of signal observed for proteins inherent in the cells, commonly called ‘‘housekeeping” (HK) proteins [34,35].”
Milani, C., Corsetto, P. A., Farina, F., Botto, L., Lonati, E., Massimino, L., Rizzo, A. M., Bulbarelli, A., & Palestini, P. (2018) · Toxicology · doi:10.1016/j.tox.2018.07.017 · Cited by 17 · see citing works
Effects of PM2.5 Exposure on the ACE/ACE2 Pathway: Possible Implication in COVID-19 Pandemic
Botto, L., Lonati, E., Russo, S., Cazzaniga, E., Bulbarelli, A., & Palestini, P. (2023) · International Journal of Environmental Research and Public Health · doi:10.3390/ijerph20054393 · Cited by 18 · see citing works
“Accordingly, samples were normalized with respect to the total amount of proteins detected by Ponceau staining, allowing a straightforward correction for lane-to-lane variation [45,47].”
MethodResultGibbons, A. S., Jeon, W. J., Scarr, E., & Dean, B. (2015) · The International Journal of Neuropsychopharmacology · doi:10.1093/ijnp/pyv118 · Cited by 17 · see citing works
“In completing these and the experiments comparing CHRM2 protein levels across psychiatric disorders, our experience agrees with the proposition that it is extremely difficult to find CNS reference proteins that, by definition, do not change levels between CNS regions, with disorders of the human CNS, or after drug tre…”
BackgroundMethodInfluentialMyo-Inositol Limits Kainic Acid-Induced Epileptogenesis in Rats
Kandashvili, M., Gamkrelidze, G. N., Tsverava, L., Lordkipanidze, T. G., Lepsveridze, E., Lagani, V., Burjanadze, M. A., Dashniani, M. G., Kokaia, M., & Solomonia, R. O. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23031198 · Cited by 17 · see citing works
“We have not normalized data with respect to any other housekeeping protein in brain tissue samples, because it cannot be guaranteed that such proteins are not affected by KA treatment [59,60].”
BackgroundPavković, Ž., Milanović, D., Ruždijić, S., Kanazir, S., & Pešić, V. (2018) · Pediatric Anesthesia · doi:10.1111/pan.13396 · Cited by 16 · see citing works
Abraham, M. J., Flemming, K., Raymond, S., Wong, A. Y. C., & Bergeron, R. (2019) · Physiological Reports · doi:10.14814/phy2.14147 · Cited by 15 · see citing works
“This was done to ensure that normalization did not rely on a single protein, but rather the total profile of protein found in the crude cellular extract (Li and Shen 2013).”
BackgroundRessaissi, A., Attia, N., Pacheco, R., Fale, P. L. V., & Serralheiro, M. L. (2019) · Journal of Functional Foods · doi:10.1016/j.jff.2019.103674 · Cited by 16 · see citing works
Current approaches and challenges in targeted absolute quantification of membrane proteins
Trötschel, C., & Poetsch, A. (2014) · PROTEOMICS · doi:10.1002/pmic.201400427 · Cited by 16 · see citing works
Lee, S. Y., & Nam, Y. K. (2016) · Fisheries and aquatic sciences · doi:10.1186/s41240-016-0022-z · Cited by 16 · see citing works
“…of recent studies have also claimed that these HKGs may not always be stable depending on aquatic species; they could be modulated by certain stimulatory treatments with variable expression levels under different physiological conditions (Li & Shen 2013; Taylor et al. 2013; Yuan et al. 2014).”
BackgroundLi, X., Huang, K., Chen, F., et al. (2016) · animal · doi:10.1017/s1751731115002748 · Cited by 15 · see citing works
“In fact, commonly used ICGs can considerably vary between species, tissue types and experimental conditions (Li and Shen, 2013).”
BackgroundTsverava, L., Lordkipanidze, T. G., Lepsveridze, E., Nozadze, M., Kikvidze, M., & Solomonia, R. O. (2016) · BioMed Research International · doi:10.1155/2016/2794096 · Cited by 15 · see citing works
Gomes, R. S., Silva, M. V. T., dos Santos, J. C., et al. (2018) · Infection and Immunity · doi:10.1128/iai.00796-17 · Cited by 15 · see citing works
Tsverava, L., Kandashvili, M., Margvelani, G., et al. (2019) · BioMed Research International · doi:10.1155/2019/4518160 · Cited by 15 · see citing works
Sánchez-Melgar, A., Albasanz, J. L., Pallàs, M., & Martı́n, M. (2020) · ACS Chemical Neuroscience · doi:10.1021/acschemneuro.0c00067 · Cited by 14 · see citing works
Biological validation of a novel process and product for quantitating western blots
Diller, T. C., Thompson, J., & Steer, B. A. (2020) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2020.12.012 · Cited by 14 · see citing works
Sulfur mustard induced nuclear translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)
Steinritz, D., Weber, J., Balszuweit, F., Thiermann, H., & Schmidt, A. (2013) · Chemico-Biological Interactions · doi:10.1016/j.cbi.2013.06.015 · Cited by 14 · see citing works
Dashniani, M. G., Burjanadze, M. A., Chkhikvishvili, N. C., Solomonia, R. O., Kandashvili, M., Naneishvili, T. L., Beselia, G. V., Kruashvili, L. B., & Chighladze, M. R. (2020) · Experimental Brain Research · doi:10.1007/s00221-020-05889-6 · Cited by 13 · see citing works
Shakova, F. M., Kirova, Y. I., Silachev, D. N., Romanova, G. A., & Morozov, S. G. (2021) · Brain Sciences · doi:10.3390/brainsci11030325 · Cited by 12 · see citing works
“Recent research shows that succinate is intensely produced by activated macrophages [102].”
BackgroundReference genes to study the sex-biased expression of genes regulating Drosophila metabolism
Groef, S. D., Lopes, M. R., Winant, M., Rosschaert, E., Wilms, T., Bolckmans, L., Calevro, F., & Callaerts, P. (2024) · Scientific Reports · doi:10.1038/s41598-024-58863-5 · Cited by 12 · see citing works
Chung-Wei, Y., Chen, C., Chou, W., Lin, H.-C., Jong, Y., Tsai, L., & Chuang, C. (2016) · PLoS ONE · doi:10.1371/journal.pone.0157426 · Cited by 12 · see citing works
“We firstly confirmed that the expression of GAPDH in each sample is equally expressed in both SMAmice and controls since several housekeeping genes have been found to be differentially expressed in some neurodegenerative diseases in some circumstances at protein level [49, 50].”
BackgroundMargvelani, G., Meparishvili, M., Tevdoradze, E., McCabe, B., & Solomonia, R. (2017) · NeuroReport · doi:10.1097/wnr.0000000000000936 · Cited by 9 · see citing works
Micro-RNAs, their target proteins, predispositions and the memory of filial imprinting
Margvelani, G., Meparishvili, M., Kiguradze, T., McCabe, B., & Solomonia, R. (2018) · Scientific Reports · doi:10.1038/s41598-018-35097-w · Cited by 11 · see citing works
Panahi, Y., Moghaddam, F. S., Ghasemi, Z., Jafari, M. H., Badv, R. S., Eskandari, M. R., & Pedram, M. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17101711 · Cited by 11 · see citing works
“Furthermore, the stability of expression for a particular HKG could also be dependent upon the tissue type/source used for RNA isolation and experimental design [10,11].”
BackgroundMitochondrial Proteome Changes in Rett Syndrome
Golubiani, G., van Agen, L., Tsverava, L., Solomonia, R. O., & Müller, M. (2023) · Biology · doi:10.3390/biology12070956 · Cited by 12 · see citing works
“Therefore, to rule out any unreliability that might arise from normalization to housekeeping proteins [35], we consistently verified proper sample loading by Ponceau S staining as well as by the calibration with the mentioned protein standards (see also [34]).”
MethodSzczurowska, E., Ergang, P., Kubová, H., Druga, R., Salaj, M., & Mareš, P. (2016) · Experimental Neurology · doi:10.1016/j.expneurol.2016.05.039 · Cited by 11 · see citing works
“…staining as an alternative to B-actin and Ponceau S, showed its clear superiority as a loading control for Western blots (Gilda and Gomez, 2013; Li and Shen, 2013), therefore, in our research we also have decided to use Stain-Free staining and total protein content as a normalization factor.”
Nerve Growth Factor Shows Biphasic Expression during Adjuvant-Induced Neurogenic Inflammation
Gujar, V., Pande, R. D., & Das, S. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25074029 · Cited by 10 · see citing works
“All steps were taken to limit the number of animals used in the experiments.”
BackgroundRichardson, T. E., Orr, M. E., Orr, T. C., Rohde, S. K., Ehrenberg, A. J., Thorn, E. L., Christie, T. D., Almazan, V. F., Afzal, R., Sanctis, C. D., et al. (2024) · Alzheimer s & Dementia · doi:10.1002/alz.14487 · Cited by 10 · see citing works
“32,33 All ROIs were re-normalized from our previous analysis 28 with the context of an additional hippocampal subregion (CA4) and an additional neuropathologic entity (CTE).”
Krishnan, V. S., White, Z. R., Terrill, J. R., Hodgetts, S. I., Fitzgerald, M., Shavlakadze, T., Harvey, A. R., & Grounds, M. D. (2017) · Biogerontology · doi:10.1007/s10522-017-9714-8 · Cited by 9 · see citing works
What's the norm in normalization? A frightening note on the use of RT-qPCR in the livestock science
Busato, S., Mezzetti, M., Logan, P., Aguilera, N., & Bionaz, M. (2018) · Gene · doi:10.1016/j.gene.2018.100003 · Cited by 9 · see citing works
“, ACTB and GAPDH, RNA18S), widespread during the days of northern blotting for their ubiquitous and strong expression, yet far from being a “gold standard” (Tricarico et al., 2002; Thellin et al., 1999; Li and Shen, 2013).”
BackgroundBaasch‐Skytte, T., Gunnarsson, T. P., Fiorenza, M., & Bangsbo, J. (2021) · Physiological Reports · doi:10.14814/phy2.14681 · Cited by 8 · see citing works
“No housekeeping proteins were used as loading controls, as this procedure may increase the variation in the determination of the primary protein of interest (Goasdoue et al., 2016; Li & Shen, 2013; Murphy & Lamb, 2013).”
MethodLi, S.-W., Chen, Y.-C. D., Sheen, J., Hsu, M., Tain, Y., Chang, K., & Huang, L. (2017) · Life Sciences · doi:10.1016/j.lfs.2017.03.023 · Cited by 8 · see citing works
Liu, T., Dai, C., Xu, J., Li, S., & Chen, J. (2020) · American Journal of Physiology-Renal Physiology · doi:10.1152/ajprenal.00381.2019 · Cited by 8 · see citing works
Andersen, J. M., Opdal, S. H., Müller, C. P., & Boix, F. (2020) · Behavioural Brain Research · doi:10.1016/j.bbr.2020.112676 · Cited by 7 · see citing works
“has already been shown that β-actin, or other “housekeeping” proteins, can be affected by the experimental conditions and therefore are not suitable as loading control [27,28].”
BackgroundZhou, C., Qiu, S., Wang, F.-M., Liu, Y.-C., Hu, W. M., Yang, M.-L., Liu, W., & Li, H. (2024) · Frontiers in Pharmacology · doi:10.3389/fphar.2024.1449452 · Cited by 8 · see citing works
“New studies suggested that in neurological diseases, housekeeping proteins such as β -actin and GAPDH may be changed after injury or in neuropathological states (Li and Shen, 2013).”
BackgroundTotal protein staining with Congo red as an alternative loading control for western blot analysis
Wang, J., Li, M., Zhang, J., et al. (2021) · Biotechnic & Histochemistry · doi:10.1080/10520295.2021.2008008 · Cited by 7 · see citing works
“Consistent with earlier reports (Welinder and Ekblad 2011; Eaton et al. 2013; Li and Shen 2013), we observed the saturation point for housekeeping proteins is < 10 μg total protein for WB analysis and only in this range can housekeeping proteins serve as good loading controls.”
ResultSoderstrom, C. I., Larsen, J., Owen, C., Gifondorwa, D. J., Beidler, D. R., Yong, F. H., Conrad, P., Neubert, H., Moore, S. A., & Hassanein, M. (2022) · The AAPS Journal · doi:10.1208/s12248-022-00776-0 · Cited by 7 · see citing works
Lu, Q., Meng, X., Yang, F., et al. (2020) · Biotechnology & Biotechnological Equipment · doi:10.1080/13102818.2020.1719020 · Cited by 7 · see citing works
Long-term effects of myo-inositol on traumatic brain injury: Epigenomic and transcriptomic studies
Oganezovi, N., Lagani, V., Kikvidze, M., Gamkrelidze, G. N., Tsverava, L., Lepsveridze, E., Kelly, K. M., & Solomonia, R. O. (2024) · IBRO Neuroscience Reports · doi:10.1016/j.ibneur.2024.01.009 · Cited by 6 · see citing works
“” We have not normalized obtained density data with respect to any other housekeeping protein in brain tissue samples because it cannot be guaranteed that such proteins are not affected by CCI e.g. (Li and Shen, 2013).”
Background
- 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 › Medicine › Physiology › 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
