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Total protein is an effective loading control for cerebrospinal fluid western blots.

ABSTRACT: Cerebrospinal fluid (CSF) has been used to identify biomarkers of neurological disease. CSF protein biomarkers identified by high-throughput methods, however, require further validation. While Western blotting (WB) is well-suited to this task, the lack of a validated loading control for CSF WB limits the method's accuracy.We investigated the use of total protein (TP) as a CSF WB loading control. Using iodine-based reversible membrane staining, we determined the linear range and consistency of the CSF TP signal. We then spiked green fluorescent protein (GFP) into CSF to create defined sample-to-sample differences in GFP levels that were measured by WB before and after TP loading correction. Levels of CSF complement C3 and cystatin C measured by WB with TP loading correction and ELISA in amyotrophic lateral sclerosis and healthy control CSF samples were then compared.CSF WB with the TP loading control accurately detected defined differences in GFP levels and corrected for simulated loading errors. Individual CSF sample Western blot and ELISA measurements of complement C3 and cystatin C were significantly correlated and the methods showed a comparable ability to detect between-groups differences.CSF TP staining has a greater linear dynamic range and sample-to-sample consistency than albumin, a commonly used CSF loading control. The method accurately corrects for simulated errors in loading and improves the sensitivity of CSF WB compared to using no loading control.The TP staining loading control improves the sensitivity and accuracy of CSF WB results.

SUBMITTER: Collins MA

PROVIDER: S-EPMC4540354 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Total protein is an effective loading control for cerebrospinal fluid western blots.

Collins Mahlon A MA An Jiyan J Peller Danielle D Bowser Robert R

Journal of neuroscience methods 20150522

<h4>Background</h4>Cerebrospinal fluid (CSF) has been used to identify biomarkers of neurological disease. CSF protein biomarkers identified by high-throughput methods, however, require further validation. While Western blotting (WB) is well-suited to this task, the lack of a validated loading control for CSF WB limits the method's accuracy.<h4>New method</h4>We investigated the use of total protein (TP) as a CSF WB loading control. Using iodine-based reversible membrane staining, we determined ...[more]

PMID: 26004848

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Project description:Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to ?-actin in these circumstances could result in 'skewing' of all data by ?20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.

Project description:Huntington's disease (HD) is a hereditary neurodegenerative condition with no therapeutic intervention known to alter disease progression, but several trials are ongoing and biomarkers of disease progression are needed. Tau is an axonal protein, often altered in neurodegeneration, and recent studies pointed out its role on HD neuropathology. Our goal was to study whether cerebrospinal fluid (CSF) tau is a biomarker of disease progression in HD. After informed consent, healthy controls, pre-symptomatic and symptomatic gene expansion carriers were recruited from two HD clinics. All participants underwent assessment with the Unified HD Rating Scale '99 (UHDRS). CSF was obtained according to a standardized lumbar puncture protocol. CSF tau was quantified using enzyme-linked immunosorbent assay. Comparisons between two groups were tested using ancova. Pearson's correlation coefficients were calculated for disease progression. Significance level was defined as p < 0.05. Seventy-six participants were included in this cross-sectional multicenter international pilot study. Age-adjusted CSF tau was significantly elevated in gene expansion carriers compared with healthy controls (p = 0.002). UHDRS total functional capacity was significantly correlated with CSF tau (r = -0.29, p = 0.004) after adjustment for age, and UHDRS total motor score was significantly correlated with CSF tau after adjustment for age (r = 0.32, p = 0.002). Several UHDRS cognitive tasks were also significantly correlated with CST total tau after age-adjustment. This study confirms that CSF tau concentrations in HD gene mutation carriers are increased compared with healthy controls and reports for the first time that CSF tau concentration is associated with phenotypic variability in HD. These conclusions strengthen the case for CSF tau as a biomarker in HD. In the era of novel targeted approaches to Huntington's disease, reliable biomarkers are needed. We quantified Tau protein, a marker of neuronal death, in cerebrospinal fluid and found it was increased in patients with Huntington's disease and predicted motor, cognitive, and functional disability in patients. It is therefore likely to be a biomarker of disease progression, and possibly of therapeutic response. Read the Editorial Highlight for this article on page 9.

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Total protein is an effective loading control for cerebrospinal fluid western blots.

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Total protein is an effective loading control for cerebrospinal fluid western blots.

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