Comparison of Stain-Free gels with traditional immunoblot loading control methodology

Colella, Alex D.; Chegenii, Nusha; Tea, Melinda N; Gibbins, Ian L.; Williams, Keryn A.; Chataway, Tim

doi:10.1016/j.ab.2012.08.015

Cited by 125 publications

(125 citation statements)

References 7 publications

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“…Stain-free imaging of the gels and membranes, corresponding to proteins in the samples, was used as loading control and for normalization. 62 Proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo system (BioRad). They were then probed with a polyclonal goat anti-apoE antibody (1 : 800; AB947, Millipore), washed, and probed with HRP-conjugated horse anti-goat IgG (1 : 3000; Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease

et al. 2014

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In the adult brain, neurons require local cholesterol production, which is supplied by astrocytes through apoE-containing lipoproteins. In Huntington's disease (HD), such cholesterol biosynthesis in the brain is severely reduced. Here we show that this defect, occurring in astrocytes, is detrimental for HD neurons. Astrocytes bearing the huntingtin protein containing increasing CAG repeats secreted less apoE-lipoprotein-bound cholesterol in the medium. Conditioned media from HD astrocytes and lipoprotein-depleted conditioned media from wild-type (wt) astrocytes were equally detrimental in a neurite outgrowth assay and did not support synaptic activity in HD neurons, compared with conditions of cholesterol supplementation or conditioned media from wt astrocytes. Molecular perturbation of cholesterol biosynthesis and efflux in astrocytes caused similarly altered astrocyteneuron cross talk, whereas enhancement of glial SREBP2 and ABCA1 function reversed the aspects of neuronal dysfunction in HD. These findings indicate that astrocyte-mediated cholesterol homeostasis could be a potential therapeutic target to ameliorate neuronal dysfunction in HD. Huntington's disease (HD) is an adult-onset neurodegenerative disorder characterized by cell loss mainly in the striatum and cortex. Its pathophysiology is linked to an expanded CAG repeat in the IT-15 gene, which leads to an elongated polyQ tract in huntingtin (HTT) protein. No disease-modifying treatment is available for HD and novel pathophysiological insights and therapeutic strategies are needed. 1 Lipids are vital to brain health and function. Accordingly, the brain has a local source of cholesterol, 2 and a breakdown of cholesterol synthesis causes brain malformations and impaired cognitive function. 3,4 Cholesterol metabolism is disrupted in HD 5,6 as revealed by transcriptional, biochemical, and mass spectrometry analyses in HD rodent models. 7,8 This dysregulation is linked to a specific action of mutant HTT on sterol-regulatory-element-binding proteins (SREBPs) and on its target genes, whose reduced transcription leads to lower brain cholesterol levels. 7 In HD humans, brain cholesterol homeostasis is affected since pre-symptomatic stages, as determined by measurement of the brain-specific cholesterol catabolite 24-S-hydroxy-cholesterol (24OHC). 9,10 However, it remains unclear how reduced brain cholesterol would become pathological for HD neurons.In adulthood, astrocytes produce cholesterol, which is secreted as a complex with apolipoprotein (apo) E lipoproteins and delivered to neurons. 11,12 Mutant HTT is expressed in glial cells, 13,14 and transgenic mice overexpressing mutant HTT in astrocytes show age-dependent neurological symptoms. 15,16 Additionally, primary astrocytes overexpressing full-length human mutant HTT show reduced mRNA levels of cholesterol biosynthetic genes, along with impaired cellular production and secretion of apoE. 8 Here we employed molecular and cellular tools to test the impact of cholesterol perturbation between astrocytes and neur...

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Section: Methodsmentioning

confidence: 99%

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease

et al. 2014

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“…This is further complicated by the fact that some detection methods, in particular enhanced chemiluminescence using x-ray film, have a very restricted linear range, and careful attention to the experimental conditions is necessary to ensure linearity. It is typically better to normalize Western blots using total protein loading as the denominator (3)(4)(5)(6)(7)(8)(9)(10)(11). To avoid potential pitfalls and with a focus on improving transparency, the JBC strongly recommends that authors describe their methods used to quantify signal intensity, how the linearity of signal intensity with antigen loading was established, and how protein loading was normalized between lanes.…”

Section: Presentation and Quantitation Of Western Blotsmentioning

confidence: 99%

Transparency Is the Key to Quality

Fosang¹,

Colbran²

2015

Journal of Biological Chemistry

102

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“…Some researchers have stained the blots with total protein stains, such as Coomassie, Flamingo Pink, Sypro Ruby, Amido Black, Ponceau S, and stain-free technology, to measure the total protein signal in each lane as loading control 16,20,13,27,1,4,12 . The total protein loading control avoids the pitfalls associated with housekeeping proteins.…”

mentioning

confidence: 99%

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

Posch

Kohn

et al. 2013

JoVE

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The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a "black box" because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as β-actin, β-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative and reliable.

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Comparison of Stain-Free gels with traditional immunoblot loading control methodology

Cited by 125 publications

References 7 publications

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease

Transparency Is the Key to Quality

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

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