High affinity immobilization of proteins using the CrAsH/TC tag

Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC) tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC) and an optimized motif (FLNCCPGCCMEP). While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters

À travers la Haute-Autriche

À TRAVERS LA HAUTE-AUTRICHE À travers la Haute-Autriche ( - ) Einband ( - ) [Abb.]: Gosau mit Dachstein / Lac de Gosau et Dachstein ( - ) [Karte]: Salzkammergut ( - ) [Abb.]: Vue de Linz du haut du belvédère François-Joseph. ([1]) [2 Abb.]: (1)Vue de Gmunden aux bords du Traunsee. (2)Ischl-les-bains en hiver. (2) [Abb.]: Le Wolfgangsee vu du chemin de fer côtier du Schafberg. (3) [Abb.]: Sommet du Schafberg. (4) [2 Abb.]: (1)Sapin ébranlé par les orages. (2)Le Mondsee. (5) [Abb.]: Hallstatt. (6) [Abb.]: Lac postérieur Langbath prés d'Ebensee. (7) [2 Abb.]: (1)Aussee. (2)Grundlsee près de Aussee. (8) [Abb.]: Hetzau et le Grand Priel. ([9]) [2 Abb.]: (1)Steyr. (2)Vallée de Windischgarsten. (10) [Abb.]: Refuge du Hofpuergl. (11) [2 Abb.]: (1)Almsee près de Gruenau. (2)Eboulis du Totengebirge. (12) [2 Abb.]: (1)Poestlingberg en hiver. (2)Linz sur le Danube. (13) [Abb.]: Jeune fille de Linz dans ses atours des dimanches. (14) [2 Abb.]: (1)Grein sur le Danube. (2)Le Danube près du "Struden". (15) [Abb.]: Vapeur sur l'Attersee. (16) [Karte]: Übersichtskarte von Oberösterreich. ( - ) [Karte]: Carte synoptique des chemins de fer. Lignes des chemins de fer en Haute-Autriche (et les lignes internationales correspondantes). ( - ) Einband ( -

Azide-tagged sphingolipids for the proteome-wide identification of C16-ceramide-binding proteins

Ceramide plays key roles in autophagy, inflammation and apoptosis. However, little is known about the molecular mechanisms regulating its function and only a handful of cellular effectors are known for this lipid. Here we show that azide-tagged sphingolipids are powerful tools to identify ceramide targets. The combination of a protein array analysis and a mass spectrometry-based proteomic profiling successfully detects known ceramide-binding proteins and identifies others not yet reported, several of which we validated using a variety of techniques. © 2018 The Royal Society of Chemistry.The Dortmund Protein Facility is acknowledged for assistance in cloning, protein expression and purification. We would like to thank Dr I. Vetter for fruitful discussion and for the generous gift of iFABP. The research leading to these results has received funding from the Max Planck Society (MPG Partner Group) and partial support from the Ministerio de Economia and Competitividad (grant CTQ2013-44334-P). G. T. thanks Prof. H. Waldmann for his constant and generous support.Peer reviewe

Site-specific, reversible and fluorescent immobilization of proteins on CrAsH-modified surfaces for microarray analytics

A novel technique for protein immobilization onto CrAsH-modified surfaces is presented. This approach enables an efficient, reversible and fluorogenic immobilization of proteins. Moreover, expressed proteins can also be directly immobilized from cellular lysates without prior purification. The immobilized proteins are suitable for protein-protein interaction studies and the fluorescence enhancement upon immobilization can be employed for the direct detection of the immobilized protein without the need for secondary detection methods.Peer reviewe