High throughput protein expression screening in the nervous system – needs and limitations

Anderson, Chris; Grant, Seth G. N.

doi:10.1113/jphysiol.2006.113795

Cited by 24 publications

(18 citation statements)

References 32 publications

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“…They demonstrate rather diverse morphological, physiological, molecular, and synaptic characteristics. According to some estimates, the total number of various types of neocortical neurons reach 10 3 and even 10 4 [12]. At the level of a local cortical neuronal network (area of the cortical region about 1 mm 2 ), the variety of types of cellular elements is, to a considerable extent, determined by heterogeneity of the population of inhibitory interneurons.…”

Section: General Structure Of the System Of Interneurons Of The Neocomentioning

confidence: 99%

Variety of types of cortical interneurons

et al. 2007

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A most important component of the mammalian neocortex is the system of inhibitory interneurons. It is composed of cellular elements, which differ from each other in morphological, electrophysiological, and genetical features; these cells form a complex system of synaptic connections with glutamatergic cells and with each other. Some regularities that characterize the variety of types of cortical interneurons are discussed in our study.

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Section: General Structure Of the System Of Interneurons Of The Neocomentioning

confidence: 99%

Variety of types of cortical interneurons

et al. 2007

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“…Relative to high-throughput technologies for assessing gene expression differences between experimental samples (e.g., microarrays, deep sequencing), comparative proteomic technologies are limited by sensitivity and reproducibility (Anderson and Grant 2006; Gutstein et al 2008). Historically, quantitative proteomic studies have typically relied on two-dimensional difference gel electrophoresis (2D-DIGE) for the separation and visualization of experimental and control samples.…”

Section: Discussionmentioning

confidence: 99%

Microproteomics: Quantitative Proteomic Profiling of Small Numbers of Laser-Captured Cells

Roulhac¹,

Ward²,

Thompson³

et al. 2011

Cold Spring Harb Protoc

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INTRODUCTIONDuring the last decade, significant progress in the analysis of whole genomes and transcriptomes has triggered efforts to analyze the proteome. Advancements in protein extraction, purification, and identification have been driven by the development of mass spectrometers with greater sensitivity and resolution. Nevertheless, comparative and quantitative proteomic technologies have not progressed to the extent of genomic and transcriptomic technologies for accessing gene expression differences. Unlike the genome, which is similar throughout all cells in a given organism, the proteome varies in different cells. Also, there is no self-replicating amplification mechanism for proteins such as the polymerase chain reaction (PCR) for DNA. Therefore, developing methods that extract, separate, detect, and identify proteins from extremely small samples are needed. The advent of laser capture microdissection (LCM) has expanded the analytical capabilities of proteomics. LCM has proven an effective technique to harvest pure cell populations from tissue sections. This protocol describes a microproteomic platform that uses nanoscale liquid chromatography/tandem mass spectrometry (nano-LC-MS/MS) to simultaneously identify and quantify hundreds of proteins from LCMs of tissue sections from small tissue samples containing as few as 1000 cells. The LCM-dissected tissues are subjected to protein extraction, reduction, alkylation, and digestion, followed by injection into a nano-LC-MS/MS system for chromatographic separation and protein identification. The approach can be validated by secondary screening using immunological techniques such as immunohistochemistry or immunoblots.

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“…In general, neuroscientists have been at the forefront in advocating for enhanced reliability and reproducibility of Ab-based research, promoting both stringent validation of Abs [2; 33; 48], and transparent, unambiguous description of those used in published research [49]. Neuroscience journals with high publication standards such as the Journal of Comparative Neurology ( JCN ) have implemented strict measures for manuscripts reporting Ab-based research [1; 50].…”

Section: Neuromab and The Outlook For The Future Of Ab Qualitymentioning

confidence: 99%

Developing high-quality mouse monoclonal antibodies for neuroscience research – approaches, perspectives and opportunities

2016

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High-quality antibodies (Abs) are critical to neuroscience research, as they remain the primary affinity proteomics reagent used to label and capture endogenously expressed protein targets in the nervous system. As in other fields, neuroscientists are frequently confronted with inaccurate and irreproducible Ab-based results and/or reporting. The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use against mammalian brain (i.e., NeuroMabs). Here we describe our methodology of multi-step mAb screening focused on identifying mAbs exhibiting efficacy and specificity in labeling mammalian brain samples. We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling. We also describe why our emphasis on extensive validation of large numbers of candidates by immunoblotting and immunohistochemistry against brain samples is essential for identifying those that exhibit efficacy and specificity in those applications to become NeuroMabs. We describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies. We detail our recent use of recombinant cloning of NeuroMabs as a method to archive all NeuroMabs, to unambiguously define NeuroMabs at the DNA sequence level, and to re-engineer IgG1 NeuroMabs to less common IgG subclasses to facilitate their use in multiplex labeling. Finally, we provide suggestions to facilitate Ab development and use, as to design, execution and interpretation of Ab-based neuroscience experiments. Reproducibility in neuroscience research will improve with enhanced Ab validation, unambiguous identification of Abs used in published experiments, and end user proficiency in Ab-based assays.

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High throughput protein expression screening in the nervous system – needs and limitations

Cited by 24 publications

References 32 publications

Variety of types of cortical interneurons

Variety of types of cortical interneurons

Microproteomics: Quantitative Proteomic Profiling of Small Numbers of Laser-Captured Cells

Developing high-quality mouse monoclonal antibodies for neuroscience research – approaches, perspectives and opportunities

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