Immobilized Peptides to Study Protein-Protein Interactions — Potential and Pitfalls

Bräuning, Rüdiger; Mähler, Michael; Hügle-Dörr, Barbara; Blüthner, Martin; Koch, Joachim; Petersen, Gabriele

doi:10.1007/978-3-662-09229-3_11

Cited by 6 publications

(2 citation statements)

References 15 publications

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“…To avoid unwanted side effects, the right choice of buffer solutions is advised. However, in the case of SPOT synthesis, even the optimal buffer composition8, 24 fails to prevent cross‐reaction. Our approach identified several amino acids that interact with different detection systems.…”

Section: Resultsmentioning

confidence: 99%

A study to assess the cross‐reactivity of cellulose membrane‐bound peptides with detection systems: an analysis at the amino acid level

Mahrenholz

Tapia

Stigler

et al. 2010

Journal of Peptide Science

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The growing demand for binding assays to study protein-protein interaction can be addressed by peptide array-based methods. The SPOT technique is a widespread peptide-array technology, which is able to distinguish semi-quantitatively the binding affinities of peptides to defined protein targets within one array. The quality of an assay system used for probing peptide arrays depends on the well-balanced combination of screening and read-out methods. The former address the steady-state of analyte capture, whereas the latter provide the means to detect captured analyte. In all cases, however, false-positive results can occur when challenging a peptide array with analyte or detecting captured analyte with label conjugates. Little is known about the cross-reactivity of peptides with the detection agents. Here, we describe at the amino acid level the potential of (i) 5-(and 6)-carboxytetramethylrhodamine (5(6)-TAMRA), (ii) fluoresceinisothiocyanate in form of the peptide-bound fluorescein-substituted thiourea derivative (FITC), and (iii) biotin/streptavidin-POD to cross-react with individual amino acids in a peptide sequence.

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Section: Resultsmentioning

confidence: 99%

A study to assess the cross‐reactivity of cellulose membrane‐bound peptides with detection systems: an analysis at the amino acid level

Mahrenholz

Tapia

Stigler

et al. 2010

Journal of Peptide Science

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“…[60][61][62] With a view to identifying the optimal conditions for IgE screening, we investigated the influence of five blocking reagents on the readout quality of the assay: bovine serum albumin (BSA), Tween 20, polymer, or milk powder-based blocking buffers and Sigma-Genosys blocking buffer concentrate in Tris-buffered saline (TBS) with sucrose (5 %, w/v). The capacity of these five blocking reagent mixtures to suppress unspecific binding was tested on a synthetic 12-meric pepscan over the Api g 1.0101 sequence prepared on a N-CAPE membrane with high loading (as above).…”

Section: Blocking Reagent and Incubation Parametersmentioning

confidence: 99%

Identification of IgE Binding to Api g 1‐Derived Peptides

et al. 2010

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Celery is a frequent cause of food allergy in pollen-sensitized patients and can induce severe allergic reactions. Clinical symptoms cannot be predicted by skin prick tests (SPTs) or by determining allergen-specific immunoglobulin E (IgE). Our aim was to identify specific IgE binding peptides by using an array technique. For our study, the sera of 21 patients with positive double-blind, placebo-controlled food challenge (DBPCFC) to celery, as well as the sera of 17 healthy patients were used. Additionally, all patients underwent skin tests along with determinations of specific IgE binding. The major allergen of celery Api g 1.0101 (Apium graveolens) was synthesized as an array of overlapping peptides and probed with the patients' sera. We developed an improved immunoassay protocol by investigating peptide lengths, peptide densities, incubation parameters, and readout systems, which could influence IgE binding. Sera of celery-allergic patients showed binding to three distinct regions of Api g 1.0101. The region including amino acids 100 to 126 of Api g 1.0101 is the most important region for IgE binding. This region caused a fivefold higher binding of IgE from the sera of celery-allergic patients compared to those of healthy individuals. In particular, one peptide (VLVPTADGGSIC) was recognized by all sera of celery-allergic patients. In contrast, no binding to this peptide was detected in sera of the healthy controls. Our improved assay strategy allows us to distinguish between celery-allergic and healthy individuals, but needs to be explored in a larger cohort of well-defined patients.

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Synthesis and Application of Peptide Arrays: Quo Vadis SPOT Technology

Volkmer

2009

ChemBioChem

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Hitting the SPOT: In 1992, Ronald Frank published the first seminal paper on simultaneous parallel synthesis of multiple peptides on filter paper. He defined the approach as SPOT synthesis, an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Here, a basic overview of this technology is presented and a recently published applications are highlighted. At the end, the future of peptide arrays is discussed.magnified image

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Immobilized Peptides to Study Protein-Protein Interactions — Potential and Pitfalls

Cited by 6 publications

References 15 publications

A study to assess the cross‐reactivity of cellulose membrane‐bound peptides with detection systems: an analysis at the amino acid level

A study to assess the cross‐reactivity of cellulose membrane‐bound peptides with detection systems: an analysis at the amino acid level

Identification of IgE Binding to Api g 1‐Derived Peptides

Synthesis and Application of Peptide Arrays: Quo Vadis SPOT Technology

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