β‐Actin is not a reliable loading control in Western blot analysis

Dittmer, Angela; Dittmer, Jürgen

doi:10.1002/elps.200500785

Cited by 155 publications

(139 citation statements)

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“…Antibodies recognizing nuclear factor of activated T cells 1 (NFAT-1), Ets1, or extracellular signal-regulated kinase 1/2 (ERK1/2) were from BD Transduction Laboratories (1:2,500), Santa Cruz Biotechnology, Inc. (C20, 1:2,000), or Cell Signaling (1:1,000), respectively. GAPDH-specific (1:5,000; Ambion) and ERK1/2-specific antibodies were used to check for equal protein loading (25). As GAPDH is also abundant in the nucleus (26), we used the GAPDHspecific antibody also to control protein loading of nuclear extracts.…”

Section: Methodsmentioning

confidence: 99%

Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

et al. 2007

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Rho GDP dissociation inhibitor B (Rho-GDIB), an inhibitor of Rho GTPases, is primarily expressed by hematopoietic cells but is also found in epithelial cancer cells. Recently, we have identified Rho-GDIB as a target of the transcription factor Ets1. Here, we show that, in breast cancer cells, Ets1 regulates Rho-GDIB expression and binds to the upstream region of the Rho-GDIb gene. Furthermore, in primary breast cancer, Rho-GDIB is coexpressed with Ets1. Studying the function of Rho-GDIB in breast cancer, we found that a Rho-GDIB-specific small interfering RNA increased cellular migration but also decreased the expression of cyclooxygenase-2 (Cox-2) oncogene as shown by microarray, quantitative reverse transcription-PCR, and Western blot analyses. Further studies revealed that Rho-GDIB regulates Cox-2 gene at least partly on the transcriptional level, most likely by activating nuclear factor of activated T cells 1 (NFAT-1). Vav-1, an interaction partner of Rho-GDIB, was also found to interfere with Cox-2 expression and NFAT-1 cellular distribution, suggesting a cooperative action of Rho-GDIB and Vav-1 on Cox-2 expression. To explore the importance of Rho-GDIB for the survival of breast cancer patients, two cohorts, including 263 and 117 patients, were analyzed for clinical outcome in relation to Rho-GDIB RNA and protein levels, respectively. Expression of Rho-GDIB was not associated with either disease-free or overall survival in the two patient population. Our data suggest that the expression of Rho-GDIB in breast cancer is neither beneficial nor disadvantageous to the patient. This may be the net effect of two opposing activities of Rho-GDIB, one that suppresses tumor progression by inhibiting migration and the other that stimulates it by enhancing Cox-2 expression. [Cancer Res 2007;67(22):10694-702]

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Section: Methodsmentioning

confidence: 99%

Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

et al. 2007

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“…The bands were quantified by mean optical density using computer-assisted densitometry with ImageJ v1.41 (National Institutes of Health, USA). As loading controls (eg, actin and GAPDH) often used for in western blot experiments are subject to quantitation errors (Aldridge et al, 2008;Dittmer and Dittmer, 2006), each gel was normalized to a total protein stain (Swift stain; G-Biosciences, St Louis, MO). Before each study, a series of western blottings were performed using different titrations of sample and antibody, to establish the linear range for each response.…”

Section: Western Blottingmentioning

confidence: 99%

Chronic Intermittent Ethanol Exposure Enhances the Excitability and Synaptic Plasticity of Lateral Orbitofrontal Cortex Neurons and Induces a Tolerance to the Acute Inhibitory Actions of Ethanol

Nimitvilai

López

Mulholland

et al. 2015

Neuropsychopharmacol

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Alcoholism is associated with changes in brain reward and control systems, including the prefrontal cortex. In prefrontal areas, the orbitofrontal cortex (OFC) has been suggested to have an important role in the development of alcohol-abuse disorders and studies from this laboratory demonstrate that OFC-mediated behaviors are impaired in alcohol-dependent animals. However, it is not known whether chronic alcohol (ethanol) exposure alters the fundamental properties of OFC neurons. In this study, mice were exposed to repeated cycles of chronic intermittent ethanol (CIE) exposure to induce dependence and whole-cell patch-clamp electrophysiology was used to examine the effects of CIE treatment on lateral OFC (lOFC) neuron excitability, synaptic transmission, and plasticity. Repeated cycles of CIE exposure and withdrawal enhanced current-evoked action potential (AP) spiking and this was accompanied by a reduction in the afterhyperpolarization and a decrease in the functional activity of SK channels. CIE mice also showed an increase in the AMPA/NMDA ratio, and this was associated with an increase in GluA1/GluA2 AMPA receptor expression and a decrease in GluN2B NMDA receptor subunits. Following CIE treatment, lOFC neurons displayed a persistent long-term potentiation of glutamatergic synaptic transmission following a spike-timing-dependent protocol. Lastly, CIE treatment diminished the inhibitory effect of acute ethanol on AP spiking of lOFC neurons and reduced expression of the GlyT1 transporter. Taken together, these results suggest that chronic exposure to ethanol leads to enhanced intrinsic excitability and glutamatergic synaptic signaling of lOFC neurons. These alterations may contribute to the impairment of OFC-dependent behaviors in alcohol-dependent individuals.

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“…Unfortunately, this discrepancy in protein abundance between POI and the LC means that homogenate concentrations that allow the POI to be in the linear range of detection on a polyacrylamide gel, necessarily put the LC outside the linear range of detection. Recently it was shown that β-actin is a poor control for many Western blot analyses because at the protein concentrations most often used, optical density values are not only outside the linear range, but they become essentially uncorrelated with protein concentration (Dittmer and Dittmer, 2006). This second issue is pertinent even in the case of qualitative studies, where loading controls are just compared visually, because such studies often assume that if the protein bands appear to be equal, they must be very nearly so.…”

Section: Introductionmentioning

confidence: 99%

The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting

Aldridge

Podrebarac

Greenough

et al. 2008

Journal of Neuroscience Methods

399

345

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Western blots are used to estimate the relative concentrations of proteins of interest based on staining by specific antibodies. Quantitative measurements are often subject to error due to overloading of the loading control and over-reliance on normalization. We have found that at the protein concentrations normally used to quantify most low-abundance proteins of interest, frequently used single-protein loading controls, such as GAPDH and β-actin, do not accurately reflect differences in protein concentration. Two total protein stains, SYPRO® Ruby and Amido Black, were compared and found to be acceptable alternatives to single protein controls. Although we cannot prove that high-abundance loading controls are inaccurate under all possible conditions, we conclude that the burden of proof should lie with the researcher to demonstrate that their loading control is reflective of quantitative differences in protein concentration.

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β‐Actin is not a reliable loading control in Western blot analysis

Cited by 155 publications

References 4 publications

Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

Cyclooxygenase-2 Is a Target Gene of Rho GDP Dissociation Inhibitor β in Breast Cancer Cells

Chronic Intermittent Ethanol Exposure Enhances the Excitability and Synaptic Plasticity of Lateral Orbitofrontal Cortex Neurons and Induces a Tolerance to the Acute Inhibitory Actions of Ethanol

The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting

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