Springer protocols feed by Immunology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.A General Method for Measuring Persister Levels in Escherichia coli Cultures
Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bactericidal antibiotics. These cells are suggested to be involved in the establishment of chronic infections. Persister levels depend on growth conditions. Here, we discuss the parameters that have to be considered when measuring persister levels and provide a sample protocol to do it. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
Persisters: Methods for Isolation and Identifying Contributing Factors—A Review
Persister cells are phenotypic variants surviving a lethal dose of antibiotic, sufficient to kill the bulk of an exponential phase population. In this chapter we summarize current techniques to isolate persisters and discuss limitations associated with identifying mechanisms of persister formation. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
A Historical Perspective on Bacterial Persistence
Bactericidal antibiotics quickly kill the majority of a bacterial population. However, a small fraction of cells typically survive through entering the so-called persister state. Persister cells are increasingly being viewed as a major cause of the recurrence of chronic infectious disease and could be an important factor in the emergence of antibiotic resistance. The phenomenon of persistence was first described in the 1940s, but remained poorly understood for decades afterwards. Only recently, a series of breakthrough discoveries has started to shed light on persister physiology and the molecular and genetic underpinnings...
Source: Springer protocols feed by Immunology - October 16, 2015 Category: Allergy & Immunology Source Type: news
The Peptide Microarray-Based Resonance Light Scattering Assay for Sensitively Detecting Intracellular Kinase Activity
The peptide microarray technology is a robust, reliable, and efficient technique for large-scale determination of enzyme activities, and high-throughput profiling of substrate/inhibitor specificities of enzymes. Here, the activities of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) in different cell lysates have been detected by a peptide microarray-based resonance light scattering (RLS) assay with gold nanoparticle (GNP) probes. Highly sensitive detection of PKA activity in 0.1 μg total cell proteins of SHG-44 (human glioma cell) cell lysate (corresponding to 200 cells) is achieved by a selected...
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes
With the increasing need for understanding antibody specificity in antibody and vaccine research, pepscan assays provide a rapid method for mapping and profiling antibody responses to continuous epitopes. We have developed a relatively low-cost method to generate peptide microarray slides for studying antibody binding. Using a setup of an IntavisAG MultiPep RS peptide synthesizer, a Digilab MicroGrid II 600 microarray printer robot, and an InnoScan 1100 AL scanner, the method allows the interrogation of up to 1536 overlapping, alanine-scanning, and mutant peptides derived from the target antigens. Each peptide is tagged wi...
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing
This report aims to provide practical advice to those who develop or use arrays with up to ~100 different peptide or protein features. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Secondary Structure Determination of Peptides and Proteins After Immobilization
The presentation of immobilized peptides and other small biomolecules attached to surfaces can be greatly affected by the attachment chemistry and linking moieties, resulting in altered activity and specificity. For this reason, it is critical to understand how the various aspects of surface immobilization—underlying substrate properties, tether structure, and site of linkage—affect the secondary and quaternary structures of the immobilized species. Here, we present methods for attaching cysteine-containing peptides to quartz surfaces and determining the secondary structure of surface-immobilized peptides. We s...
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Analysis of Protein Tyrosine Kinase Specificity Using Positional Scanning Peptide Microarrays
We describe a peptide microarray approach to rapidly determine tyrosine kinase phosphorylation site motifs. This method uses a peptide library that systematically substitutes each of the amino acid residues at multiple positions surrounding a central tyrosine residue. Peptide substrates are synthesized as biotin conjugates for immobilization on avidin-coated slides. Following incubation of the slide with protein kinase and radiolabeled ATP, the relative extent of phosphorylation of each of the peptides is quantified by phosphor imaging. This method allows small quantities of kinase to be analyzed rapidly in parallel, facil...
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
High-Throughput Microarray Incubations Using Multi-Well Chambers
Peptide microarrays are ideal tools for a variety of applications ranging from epitope mapping to immune monitoring. Here we present a method for high-throughput screening of biological samples using only standard microtiter plate equipment. Parallel incubation of a large number of samples with a small library of peptides is enabled by printing multiple identical mini-arrays on one microarray slide and further combining four slides to yield an incubation frame possessing the dimensions of a 96-well microtiter plate. Applying conventional lab equipment such as ELISA washers, hundreds of samples can be processed in 1 day yie...
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Peptide Arrays on Planar Supports
On a past volume of this monograph we have reviewed general aspects of the varied technologies available to generate peptide arrays. Hallmarks in the development of the technology and a main sketch of preparative steps and applications in binding assays were used to walk the reader through details of peptide arrays. In this occasion, we resume from that work and bring in some considerations on quantitative evaluation of measurements as well as on selected reports applying the technology. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Analyzing Peptide Microarray Data with the R pepStat Package
In this chapter we demonstrate the use of R Bioconductor packages pepStat and Pviz on a set of paired peptide microarrays generated from vaccine trial data. Data import, background correction, normalization, and summarization techniques are presented. We introduce a sliding mean method for amplifying signal and reducing noise in the data, and show the value of gathering paired samples from subjects. Useful visual summaries are presented, and we introduce a simple method for setting a decision rule for subject/peptide responses that can be used with a set of control peptides or placebo subjects. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - September 29, 2015 Category: Allergy & Immunology Source Type: news
Evaluating Cytoplasmic and Nuclear Levels of Inflammatory Cytokines in Cancer Cells by Western Blotting
Increased expression and cellular release of inflammatory cytokines, interleukin-8 (IL-8; CXCL8), and high mobility group box-1 (HMGB1) are associated with increased cell proliferation, angiogenesis, and metastasis during cancer progression. In prostate and ovarian cancer cells, increased levels of IL-8 and HMGB1 correlate with poor prognosis. We have recently shown that proteasome inhibition by bortezomib (BZ) specifically increases IL-8 release from metastatic prostate and ovarian cancer cells. In this chapter, we describe a protocol to analyze the cytoplasmic and nuclear levels of IL-8 and HMGB1 in prostate and ovarian ...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Immunofluorescence and Subsequent Confocal Microscopy of Intracellular TNF in Human Neutrophils
Immunofluorescence is an important technique required to observe expression, localization and colocalization of proteins within the cell. Here we describe the immunofluorescence and subsequent confocal microscopy technique of tumor necrosis factor-α (TNF) in human neutrophils (polymorphonuclear leukocytes; PMN). The qualitative technique can be used to observe the expression pattern changes from resting to stimulated leukocytes. Colocalization with other cytokines, proteins, or organelles can be observed. This immunofluorescence technique can be done in 1–2 days. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Interleukin-1 (IL-1) Immunohistochemistry Assay in Oral Squamous Cell Carcinoma
This chapter describes an immunohistochemistry method to analyze interleukin-1 (IL-1) in oral squamous cell carcinoma. The described protocol has been optimized for IL-1 detection in formalin-fixed paraffin-embedded oral tissue sections by light microscopy. A few common pitfalls and problems associated with immunohistochemical staining are discussed. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
Analysis of IL-17 Production by Flow Cytometry and ELISPOT Assays
Interleukin (IL)-17 represents a family of cytokines with six members, namely IL-17A, B, C, D, E, and F. IL-17A and IL-17F are best studied proinflammatory cytokines. CD4+ T helper cells producing IL-17A have been identified as a distinct T helper subset, Th17 cells. IL-17 and Th17 cells are important mediators in tissue inflammation in immune-mediated inflammatory diseases. IL-17 is also produced by other immune cells and plays an important role in host defense against microbial infection. Cell-based assays are sensitive and quantitative, and enable identification of cellular sources of IL-17 production. This chapter desc...
Source: Springer protocols feed by Immunology - June 10, 2014 Category: Allergy & Immunology Source Type: news
