Antibody microarray‐based profiling of complex specimens: systematic evaluation of labeling strategies

Kusnezow, Wlad; Banzon, Virryan; Schröder, Christoph; Schaal, René; Hoheisel, Jörg D.; Rüffer, Sven; Luft, Petra; Duschl, Albert; Syagailo, Yana V.

doi:10.1002/pmic.200600762

Cited by 64 publications

(42 citation statements)

References 37 publications

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“…The two fractions were centrifuged at 10,000 ϫ g for 10 min and the supernatants, typically containing 5-10 and 1-4 mg/ml of protein, respectively, were labeled with 0.5 mg/ml biotin-PEO 4 -NHS (Pierce) at 22°C for 20 min. This label was recently found in two studies to be optimal for detection of cytokines (10,15). Free biotin was removed by passing the proteins over a G50F spin column equilibrated with 140 mM NaCl, 40 mM HEPES, pH 7.4, and 0.1% Tween 20.…”

Section: Methodsmentioning

confidence: 99%

Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Slåstad

Carrillo

et al. 2009

Molecular & Cellular Proteomics

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Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670 -10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclindependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Molecular & Cellular Proteomics 8:245-257, 2009.

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Section: Methodsmentioning

confidence: 99%

Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Slåstad

Carrillo

et al. 2009

Molecular & Cellular Proteomics

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“…Hence, most of the targeted analytes could also be successfully detected in directly labelled, crude serum samples, representing one of the most common clinical sample format. In comparison, LODs in the pM to fM range have been observed for high-end conventional antibody microarrays [5,11,41,44,53] (Wingren et al unpublished observations), but these set-ups have then been subjected to significant optimization and method refinement.…”

Section: Discussionmentioning

confidence: 99%

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

et al. 2014

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Miniaturized (Ø 10 μm), multiplexed (>5-plex), and high-density (>100 000 spots cm−2) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, BioplumeTM—consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels—to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced—based on miniaturized spot features (78.5 um2, Ø 10 μm) at a 7–125-times increased spot density (250 000 spots cm−2), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.

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“…The detection limit was similar to that reported previously. 10,11 Data Analysis. Results are presented as means ((SD) unless otherwise specified.…”

Section: Alhamdani Et Almentioning

confidence: 99%

Single-Step Procedure for the Isolation of Proteins at Near-Native Conditions from Mammalian Tissue for Proteomic Analysis on Antibody Microarrays

et al. 2010

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The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While antibody microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.

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Antibody microarray‐based profiling of complex specimens: systematic evaluation of labeling strategies

Cited by 64 publications

References 37 publications

Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer

Single-Step Procedure for the Isolation of Proteins at Near-Native Conditions from Mammalian Tissue for Proteomic Analysis on Antibody Microarrays

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