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1.  In silico to in vivo splicing analysis using splicing code models 
Methods (San Diego, Calif.)  2013;67(1):3-12.
With the growing appreciation of RNA splicing’s role in gene regulation, development, and disease, researchers from diverse fields find themselves investigating exons of interest. Commonly, researchers are interested in knowing if an exon is alternatively spliced, if it is differentially included in specific tissues or in developmental stages, and what regulatory elements control its inclusion. An important step towards the ability to perform such analysis in silico was made with the development of computational splicing code models. Aimed as a practical how-to guide, we demonstrate how researchers can now use these code models to analyze a gene of interest, focusing on Bin1 as a case study. Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein known to be functionally regulated through alternative splicing in a tissue-specific manner. Specific Bin1 isoforms have been associated with muscular diseases and cancers, making the study of its splicing regulation of wide interest. Using AVISPA, a recently released web tool based on splicing code models, we show that many Bin1 tissue-dependent isoforms are correctly predicted, along with many of its known regulators. We review the best practices and constraints of using the tool, demonstrate how AVISPA is used to generate high confidence novel regulatory hypotheses, and experimentally validate predicted regulators of Bin1 alternative splicing.
PMCID: PMC4321995  PMID: 24321485
Alternative splicing; Splicing code; BIN1; AVISPA; Centronuclear myopathy (CNM); Myotonic dystrophy (DM)
2.  [No title available] 
PMCID: PMC3932149  PMID: 23974071
3.  [No title available] 
PMCID: PMC3943816  PMID: 24096052
4.  pCAP-Based Peptide Substrates: The New Tool in the Box of Tyrosine Phosphatase Assays 
Methods (San Diego, Calif.)  2013;65(2):165-174.
Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.
PMCID: PMC3899110  PMID: 23886911
fluorogenic enzyme substrates; peptide synthesis; high-throughput screening; enzyme activity gel; multiply phosphorylated peptides
5.  Analysis of PTP1B Sumoylation 
Methods (San Diego, Calif.)  2013;65(2):201-206.
PMCID: PMC3946832  PMID: 24076082
signal transduction; protein tyrosine phosphatases; phosphorylation; posttranslational modification; sumoylation
6.  Integrating Virtual and Biochemical Screening for Protein Tyrosine Phosphatase Inhibitor Discovery 
Methods (San Diego, Calif.)  2013;65(2):219-228.
Protein tyrosine phosphatases (PTPs) represent an important class of enzymes that mediate signal transduction and control diverse aspects of cell behavior. The importance of their activity is exemplified by their significant contribution to disease etiology with over half of all human PTP genes implicated in at least one disease. Small molecule inhibitors targeting individual PTPs are important biological tools, and are needed to fully characterize the function of these enzymes. Moreover, potent and selective PTP inhibitors hold the promise to transform the treatment of many diseases. While numerous methods exist to develop PTP-directed small molecules, we have found that complimentary use of both virtual (in silico) and biochemical (in vitro) screening approaches expedite compound identification and drug development. Here, we summarize methods pertinent to our work and others. Focusing on specific challenges and successes we have experienced, we discuss the considerable caution that must be taken to avoid enrichment of inhibitors that function by non-selective oxidation. We also discuss the utility of using “open” PTP structures to identify active-site directed compounds, a rather unconventional choice for virtual screening. When integrated closely, virtual and biochemical screening can be used in a productive workflow to identify small molecules targeting PTPs.
PMCID: PMC3946838  PMID: 23969317
protein tyrosine phosphatase; PTP; PTPσ; inhibitor; virtual screen; small molecule
7.  Analysis pipelines and packages for Infinium HumanMethylation450 BeadChip (450k) data 
The Illumina HumanMethylation450 BeadChip has become a popular platform for interrogating DNA methylation in epigenome-wide association studies (EWAS) and related projects as well as resource efforts such as the International Cancer Genome Consortium (ICGC) and the International Human Epigenome Consortium (IHEC). This has resulted in an exponential increase of 450k data in recent years and triggered the development of numerous integrated analysis pipelines and stand-alone packages. This review will introduce and discuss the currently most popular pipelines and packages and is particularly aimed at new 450k users.
PMCID: PMC4304832  PMID: 25233806
HumanMethylation450; Epigenetics; Analysis pipelines; EWAS; DNA methylation; 450k BeadChip
8.  Probe Lasso: A novel method to rope in differentially methylated regions with 450K DNA methylation data 
The speed and resolution at which we can scour the genome for DNA methylation changes has improved immeasurably in the last 10 years and the advent of the Illumina 450K BeadChip has made epigenome-wide association studies (EWAS) a reality. The resulting datasets are conveniently formatted to allow easy alignment of significant hits to genes and genetic features, however; methods that parse significant hits into discreet differentially methylated regions (DMRs) remain a challenge to implement. In this paper we present details of a novel DMR caller, the Probe Lasso: a flexible window based approach that gathers neighbouring significant-signals to define clear DMR boundaries for subsequent in-depth analysis. The method is implemented in the R package ChAMP (Morris et al., 2014) and returns sets of DMRs according to user-tuned levels of probe filtering (e.g., inclusion of sex chromosomes, polymorphisms) and probe-lasso size distribution. Using a sub-sample of colon cancer- and healthy colon-samples from TCGA we show that Probe Lasso shifts DMR calling away from just probe-dense regions, and calls a range of DMR sizes ranging from tens-of-bases to tens-of-kilobases in scale. Moreover, using TCGA data we show that Probe Lasso leverages more information from the array and highlights a potential role of hypomethylated transcription factor binding motifs not discoverable using a basic, fixed-window approach.
PMCID: PMC4304833  PMID: 25461817
Differentially methylated regions; DNA methylation; Epigenetics; EWAS; Illumina 450K BeadChip
9.  oxBS-450K: A method for analysing hydroxymethylation using 450K BeadChips 
•A method is presented for 5hmC detection and analysis using Infinium 450K BeadChips.•The oxBS-450K method can discriminate between 5mC and 5hmC in human gDNA•5hmC levels were quantified genome-wide in 3 distinct biological samples.•The reported 5hmC signal was validated using mass spectrometry and pyrosequencing.•The effects of differing amounts of input DNA on final 5hmC call rate are discussed.
DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R2 > 0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.
PMCID: PMC4304834  PMID: 25175075
Hydroxymethylation; DNA methylation; 450K BeadChip; Oxidation; Bisulfite conversion; Epigenetics
10.  Humanized FcRn Mouse Models for Evaluating Pharmacokinetics of human IgG Antibodies 
Methods (San Diego, Calif.)  2013;65(1):148-153.
A key element for the successful development of novel therapeutic antibodies is to fully understand their pharmacokinetic and pharmacodynamic behavior before performing clinical trials. While many in vitro modeling approaches exist, these simply cannot substitute for data obtained from appropriate animal models. It was established quite early that the unusual long serum half-life of immunoglobulin G’s (IgGs) and Fc domains are due to their rescue and recycling by the neonatal Fc receptor (FcRn). The diverse roles of FcRn became apparent after isolation and cloning. Interesting are the significant species differences between rodent and human FcRn reactivity, rendering wild type rodents an inadequate model for studying IgG serum half-life. With the advance of genetic engineering mouse models have been established expressing human FcRn, and lacking mouse FcRn protein. These models have become highly relevant tools for serum half-life analysis of Fc-containing compounds.
PMCID: PMC3858440  PMID: 23867339
neonatal Fc receptor; humanized mice; monoclonal antibody; serum half-life; pharmacokinetics; efficacy
11.  Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography 
Methods (San Diego, Calif.)  2013;65(1):127-132.
The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiply roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1M Citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6 mg of SIgA2 b12 was achieved from the combination of 20 mg of purified dIgA2 b12 and 2 L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a simplified means of generating a variety of pathogen-specific SIgA antibodies for research and clinical applications.
PMCID: PMC3858571  PMID: 23811333
IgA; Secretory IgA; mucosal IgA; affinity chromatography; antibody
12.  Antibody conjugation via one and two C-terminal selenocysteines 
Methods (San Diego, Calif.)  2013;65(1):133-138.
Conventional antibody conjugation methods generate antibody-drug conjugates that are heterogeneous mixtures with undefined stoichiometry and variable pharmacokinetic and pharmacodynamic properties. We have previously described a strategy to generate site-specific antibody conjugates by genetic engineering of an antibody with a single C-terminal selenocysteine, the 21st natural amino acid, which displays unique chemical reactivity allowing selective conjugation in the presence of all other natural amino acids. In the present work, we describe a method for expanding this technology to higher drug-to-antibody ratios by genetically engineering an antibody with two C-terminal selenocysteines. Both selenocysteines effectively conjugate to a fluorescent iodoacetamide derivative and the resulting conjugate fully retains its antigen binding capability. Our method provides a platform for creating stoichiometrically defined antibody-drug conjugates for therapeutic intervention.
PMCID: PMC3871924  PMID: 23756202
Antibody engineering; Antibody conjugation; Selenocysteine
13.  Engineered antibodies for molecular imaging of cancer 
Methods (San Diego, Calif.)  2013;65(1):139-147.
Antibody technology has transformed drug development, providing robust approaches to producing highly targeted and active therapeutics that can routinely be advanced through clinical evaluation and registration. In parallel, there is an emerging need to access similarly targeted agents for diagnostic purposes, including non-invasive imaging in preclinical models and patients. Antibody engineering enables modification of key properties (immunogenicity, valency, biological inertness, pharmacokinetics, clearance route, site-specific conjugation) in order to produce targeting agents optimized for molecular imaging. Expanded availability of positron-emitting radionuclides has led to a resurgence of interest and applications of immunoPET (immuno-positron emission tomography). Molecular imaging using engineered antibodies and fragments provides a general approach for assessing cell surface phenotype in vivo and stands to play an increasingly important role in cancer diagnosis, treatment selection, and monitoring of molecularly targeted therapeutics.
PMCID: PMC3947235  PMID: 24091005
engineered antibody fragments; radiolabeling; positron emission tomography; immunoPET; site-specific conjugation
14.  Single Cell Spectroscopy: Noninvasive Measures of Small-Scale Structure and Function 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.07.025.
The advancement of spectroscopy methods attained through increases in sensitivity, and often with the coupling of complementary techniques, has enabled real-time structure and function measurements of single cells. The purpose of this review is to illustrate, in light of advances, the strengths and the weaknesses of these methods. Included also is an assessment of the impact of the experimental setup and conditions of each method on cellular function and integrity. A particular emphasis is placed on noninvasive and nondestructive techniques for achieving single cell detection, including nuclear magnetic resonance, in addition to physical, optical, and vibrational methods.
PMCID: PMC3833896  PMID: 23886910
Spectroscopy; Single Cells; Nuclear Magnetic Resonance; Fluorescence Microscopy; Atomic Force Microscopy
15.  Development of a method to isolate circulating tumor cells using mesenchymal-based capture 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.06.034.
Epithelial tumor cells can become mesenchymal cells and vice versa via phenotypic transitions, a process known as epithelial plasticity. We postulate that during the process of metastasis, circulating tumor cells (CTCs) lose their epithelial phenotype and acquire a mesenchymal phenotype that may not be sufficiently captured by existing epithelial-based CTC technologies. We report here on the development of a novel CTC capture method, based on the biology of epithelial plasticity, which isolates cells based on OB-cadherin cell surface expression. Using this mesenchymal-based assay, OB-cadherin cellular events are detectable in men with metastatic prostate cancer and are less common in healthy volunteers. This method may complement existing epithelial-based methods and may be particularly useful in patients with bone metastases.
PMCID: PMC3833983  PMID: 23845299
Circulating tumor cells; epithelial plasticity; epithelial-mesenchymal transition; OB-cadherin; osteomimicry; prostate cancer
16.  Detection and isolation of auto-reactive human antibodies from primary B cells 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.06.018.
The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (~105), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.
PMCID: PMC3836837  PMID: 23811296
17.  Binding and isolation of tumor cells in biological media with perfluorocarbon microbubbles 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.08.008.
With the emerging interest in personalized medicine, there is strong demand for new technologies for clinical sample interrogation. Exfoliated tumor cells in variety of pathological samples (e.g., blood, bone marrow, urine) could provide invaluable information for diagnosis and prognosis of cancers. Here we describe a detailed method for capture and isolation of tumor cells in medium, blood, or large-volume buffy coat using EpCAM-targeted buoyant microbubbles (MBs). Perflorohexane gas lipid shell MBs were prepared with emulsification method and conjugated with antibody as described by us before (Shi et al,. PLoS One, 2013). The binding of EpCAM-targeted MBs to A549 (human lung carcinoma) and 4T1 (mouse breast carcinoma) cells spiked into BSA/PBS or blood was more than 90%, which was comparable with commercial anti-EpCAM immunomagnetic beads (DynaBeads). Anti-EpCAM MBs efficiently (75–82%) isolated BxPC3 pancreatic tumor cells spiked into medium, blood or a buffy coat, within 15–30 min of incubation. We discuss MB parameters and experimental conditions critical to achieve efficient cells binding and isolation. In conclusion, MB-assisted cell isolation is a promising method for rapid enrichment of cells and biomarkers from biological samples.
PMCID: PMC3841068  PMID: 23974072
18.  A negative selection methodology using a microfluidic platform for the isolation and enumeration of circulating tumor cells 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.05.027.
Circulating tumor cells (CTCs) exist in the peripheral blood stream of metastatic cancer patients at rates of approximately 1 CTC per billion background cells. In order to capture and analyze this rare cell population, various techniques exist that range from antibody-based surface marker positive selection to methods that use physical properties of CTCs to negatively exclude background cells from a CTC population. However, methods to capture cells for functional downstream analyses are limited due to inaccessibility of the captured sample or labeling techniques that may be prohibitive to cell function. Here, we present a negative selection method that leverages a microfluidic cell concentrator (MCC) to allow collection and analysis of this rare cell population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the MCC is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population. The ability of the MCC for patient sample processing of CTCs for enumeration was demonstrated with five patient samples, revealing an average of 0.31 CTCs per mL. The technique was compared to a previously published method – the ELISPOT – that showed similar CTC levels among the five patient samples tested. Because the MCC method does not use positive selection, the method can be applied across a variety of tumor types with no changes to the process.
PMCID: PMC3858973  PMID: 23806645
19.  Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.09.006.
A variety of enrichment/isolation technologies exist for the characterization of rare cells in the blood of cancer patients. In this article, a negative depletion process is presented and discussed which consists of red blood cell (RBC) lysis and the subsequent removal of CD45 expressing cells through immunomagnetic depletion. Using this optimized assembly on 120 whole blood specimens, from 71 metastatic breast cancer patients, after RBC lysis, the average nucleated cell log depletion was 2.56 with a 77% recovery of the nucleated cells. The necessity of exploring different anti-CD45 antibody clones to label CD45 expressing cells in this enrichment scheme is also presented and discussed. An optimized, four-color immunofluorescence staining is conducted on the cells retained after the CD45-based immunomagnetic depletion process. Different types of rare non-hematopoietic cells are found in these enriched peripheral blood samples and a wide range of external and internal markers have been characterized, which demonstrates the range and heterogeneity of the rare cells.
PMCID: PMC3874448  PMID: 24056212
Circulating tumor cells; Flow cytometry; Immunocytochemistry; Immunofluorescence; Immunomagnetic separation; Tumor-related cell marker
20.  Modelling multi-protein complexes using PELDOR distance measurements for rigid body minimisation experiments using XPLOR-NIH 
Methods (San Diego, Calif.)  2014;70(2-3):139-153.
Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model.
PMCID: PMC4274318  PMID: 25448300
Cys, cysteine; Glu, glutamic acid; Tyr, tyrosine; Vps75, vacuolar protein sorting protein 75; PELDOR; DEER; XPLOR-NIH; MTSSLwizard; Chromatin; Vps75
21.  Nanoimaging methods for biomedicine 
Methods (San Diego, Calif.)  2013;60(2):111-112.
PMCID: PMC4227392  PMID: 23721609
22.  Stable isotope imaging of biological samples with high resolution secondary ion mass spectrometry and complementary techniques 
Methods (San Diego, Calif.)  2014;68(2):317-324.
Stable isotopes are ideal labels for studying biological processes because they have little or no effect on the biochemical properties of target molecules. The NanoSIMS is a tool that can image the distribution of stable isotope labels with up to 50 nm spatial resolution and with good quantitation. This combination of features has enabled several groups to undertake significant experiments on biological problems in the last decade. Combining the NanoSIMS with other imaging techniques also enables us to obtain not only chemical information but also the structural information needed to understand biological processes. This article describes the methodologies that we have developed to correlate atomic force microscopy and backscattered electron imaging with NanoSIMS experiments to illustrate the imaging of stable isotopes at molecular, cellular, and tissue scales. Our studies make it possible to address 3 biological problems: (1) the interaction of antimicrobial peptides with membranes; (2) glutamine metabolism in cancer cells; and (3) lipoprotein interactions in different tissues.
PMCID: PMC4222523  PMID: 24556558
Stable isotope; NanoSIMS; Atomic force microscopy; Backscattered electron imaging; Correlative analysis
23.  Methods to detect replication-dependent and replication-independent DNA structure-induced genetic instability 
Methods (San Diego, Calif.)  2013;64(1):10.1016/j.ymeth.2013.08.004.
DNA can adopt a variety of alternative secondary (i.e., non-B DNA) conformations that play important roles in cellular metabolism, including genetic instability, disease etiology, and evolution. While we still have much to learn, research in this field has expanded dramatically in the past decade. We have summarized in our previous Methods review (Wang et al., Methods, 2009) some commonly used techniques to determine non-B DNA structural conformations and non-B DNA-induced genetic instability in prokaryotes and eukaryotes. Since that time, we and others have further characterized mechanisms involved in DNA structure-induced mutagenesis and have proposed both replication-dependent and replication-independent models. Thus, in this review, we highlight some current methodologies to identify DNA replication-related and replication-independent mutations occurring at non-B DNA regions to allow for a better understanding of the mechanisms underlying DNA structure-induced genetic instability. We also describe a new web-based search engine to identify potential intramolecular triplex (H-DNA) and left-handed Z-DNA-forming motifs in entire genomes or at selected sequences of interest.
PMCID: PMC3842213  PMID: 23954565
DNA structure; Genetic instability; Mutation; Replication; 2-D gel electrophoresis; Search engine
24.  An HPLC-tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products 
Methods (San Diego, Calif.)  2013;64(1):10.1016/j.ymeth.2013.07.020.
DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3mA) and N7-methylguanine (7mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described.
PMCID: PMC3812247  PMID: 23876937
base excision repair; DNA glycosylase; alkylation; methylpurine; mass spectrometry
25.  Antibody validation of immunohistochemistry for biomarker discovery: Recommendations of a consortium of academic and pharmaceutical based histopathology researchers 
Methods (San Diego, Calif.)  2014;70(1):34-38.
As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1–3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.
PMCID: PMC4240800  PMID: 24525140
Antibody validation; Immunohistochemistry; Biomarker; Guidelines; Histopathology

Results 1-25 (457)