An old method facing a new challenge: Re-visiting housekeeping proteins as internal reference control for neuroscience research

“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).”

“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).”

“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).”

“…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…”

“…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).”

“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.”

“…(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…”

“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.”

“by interaction between VHL and hypoxia-induced factor-α gene (Ferguson et al., 2005; Lu et al., 2002).”

“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…”

“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).”

“, 2009) or proliferation status, and age (Lowe et al., 2000).”

“…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…”

“…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).”

“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).”

“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).”

“…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.,…”

“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).”

“, 2011) Unstable in adipose tissue (Pérez-Pérez et al., 2012)”

“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).”

“…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.”

“Tissue-specific condition ↓ With age in leukocytes (Yu et al., 2011) Unstable in adipose tissue (Pérez-Pérez et al.”

“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”

“…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.”

“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.”

“Studies showed that both staining have detection sensitivity around 50 ng/protein band (Harper and Speicher, 2001; Ren et al., 2012).”

“Protein quantification was performed by normalizing to total protein concentration (Li & Shen, 2013; Moritz, 2017; Supporting Information Figure S8).”

“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].”

“Other common loading controls (e.g., pan-actin and b-tubulin) have also displayed poor linear ranges in the same manner (Li & Shen, 2013).”

“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”