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

Collins, Mahlon A; An, Jiyan; Peller, Danielle; Bowser, Robert

doi:10.1016/j.jneumeth.2015.05.011

Cited by 50 publications

(45 citation statements)

References 62 publications

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“…Proteins selected for our SVM classifier were validated using Western blotting (WB). Polyacrylamide gel electrophoresis (PAGE), electrophoretic transfer to PVDF membrane, and WB were performed as previously described 68 . Briefly, equal volumes of CSF were loaded in each SDS-PAGE gel lane and total protein by reversible PVDF membrane stain (G-Biosciences; St. Louis, MO, USA) was used as a loading control.…”

Section: Methodsmentioning

confidence: 99%

Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis

Collins

Hood

et al. 2015

J. Proteome Res.

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Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1,712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3 were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

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Section: Methodsmentioning

confidence: 99%

Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis

Collins

Hood

et al. 2015

J. Proteome Res.

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“…By contrast, the variation of levels of β‐actin across all subjects was ∼17% higher than that for the QCC, suggesting individual variances between donors. Our data therefore suggests that levels of β‐actin, as a proportion of total protein, is not constant in every individual and β‐actin fails a core criterion for a reference protein, which is to be present in all tissues from all individuals at a constant proportion of total tissue protein .…”

Section: Discussionmentioning

confidence: 78%

“…One critical characteristic of a reference or loading protein is that it should show no variation as a proportion of total protein between individual cases . In this study we used a quality control homogenate (the QCC) to gain an estimate of the intra‐ and inter‐gel variation for Ponceau S staining and the intensity of the β‐actin signalling.…”

Section: Discussionmentioning

confidence: 99%

“…The practice of normalising data to a reference protein, also known as a loading control, is now a common practice in Western blotting . Different to the definition of reference genes, a reference protein must be present at a constant proportion of the total protein in a tissue . In addition, similar to the criteria for reference genes, reference proteins must not be affected by variables such as disease pathophysiology, gender or drug treatments .…”

Section: Introductionmentioning

confidence: 99%

“…Different to the definition of reference genes, a reference protein must be present at a constant proportion of the total protein in a tissue . In addition, similar to the criteria for reference genes, reference proteins must not be affected by variables such as disease pathophysiology, gender or drug treatments . The validity of the concept of a reference protein has become particularly important as it has been stated that “To obtain any reliable information about the expression levels of proteins on western blots, it is essential that an appropriate loading control is used.…”

Section: Introductionmentioning

confidence: 99%

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Β‐actin does not show the characteristics of a reference protein in human cortex

et al. 2018

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Levels of a reference protein must be the same as a proportion of total protein in all tissues and, in the study of human diseases, cannot vary with factors such as age, gender or disease pathophysiology. It is increasingly apparent that there may be few, if any, proteins that display the characteristics of a reference protein within the human central nervous system (CNS). To begin to challenge this hypothesis, we used Western blotting to compare variance in levels of the “gold standard” reference protein, β‐actin, in Brodmann's area 9 from 194 subjects to variance of total transferred protein measured as intensity of Ponceau S staining. The coefficient of variance of sum intensity measurements for β‐actin levels across all donors was 47% compared to 24 and 27% for the sum intensity of Ponceau S staining measured using two different detection techniques. These data strongly suggest that the level of β‐actin, proportional to total protein, is not constant in human cortex which raises further doubt about the use of reference proteins to normalise data in human CNS studies. Considering our data, we suggest an alternative approach to presenting data from Western blotting of human CNS.

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State‐of‐the‐art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome

Lee

Hong

et al. 2016

Proteomics

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Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β-tubulin and β-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.

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

Cited by 50 publications

References 62 publications

Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis

Label-Free LC–MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis

Β‐actin does not show the characteristics of a reference protein in human cortex

State‐of‐the‐art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome

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