Conductometric Monitoring of Protein–Protein Interactions

Spera, Rosanna; Festa, Fernanda; Bragazzi, Nicola Luigi; Pechkova, Eugenia; LaBaer, Joshua; Nicolini, Claudio

doi:10.1021/pr400445v

Cited by 26 publications

(56 citation statements)

References 55 publications

(120 reference statements)

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“…In our analysis we introduced also a "normalized D factor", D N , that we defined as the ratio between the half-width halfmaximum (Γ) and the half value of the maximum value of the conductance (G max ) of the measured conductance curves [11]:…”

Section: Qcm_d Conductometermentioning

confidence: 99%

“…This technology relies upon the precise, non-contact printing capabilities of a piezoelectric inkjet printer. When compared to so-called cell-free protein arrays [7,8] can be coupled with label-free approaches: which offer unique advantages in the study of human proteome [9][10][11].…”

Section: Activepipette Piezoelectric Inkjet Technologymentioning

confidence: 99%

“…In this manuscript, we report and discuss some preliminary results of protein expression of genes related to cancer, coupling Nucleic Acid Programmable Protein Array (NAPPA) with a recently improved nanogravimetric apparatus which exploits the quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies [11,12] and using a newly developed piezoelectric liquid dispensing technology.…”

Section: Activepipette Piezoelectric Inkjet Technologymentioning

confidence: 99%

“…The QC expressing proteins consisted of 9.5 MHz, AT-cut quartz crystal of 14 mm blank diameter and 7.5 mm electrode diameter, produced by ICM (Oklahoma City, USA). The electrode material was 100Å Cr and 1000Å Au and the quartz was embedded into glass-like structures for easy handling [11,30].…”

Section: Nappa Experimentsmentioning

confidence: 99%

“…Gene expression was performed immediately before the assay, following the protocol described in [11]. Briefly, IVTT was performed using HeLa lysate mix (1-Step Human Coupled IVTT Kit, Thermo Fisher Scientific Inc.), prepared according to the manufacturers' instructions.…”

Section: Nappa Experimentsmentioning

confidence: 99%

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Nanoprobe NAPPA Arrays for the Nanoconductimetric Analysis of Ultra-Low-Volume Protein Samples Using Piezoelectric Liquid Dispensing Technology

Pechkova¹,

Wiktor²,

Bragazzi³

et al. 2015

NanoWorld J

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In the last years, the evolution and the advances of the nanobiotechnologies applied to the systematic study of proteins, namely proteomics, both structural and functional, and specifically the development of more sophisticated and largescale protein arrays, have enabled scientists to investigate protein interactions and functions with an unforeseeable precision and wealth of details. Here, we present a further advancement of our previously introduced and described Nucleic Acid Programmable Protein Arrays (NAPPA)-based nanoconductometric sensor. We coupled Quartz Crystal Microbalance with Dissipation factor Monitoring (QCM_D) with piezoelectric inkjet printing technology (namely, the newly developed ActivePipette), which enables to significantly reduce the volume of probe required for genes/proteins arrays. We performed a negative control (with master mix, or MM) and a positive control (MM_p53 plus MDM2). We performed this experiment both in static and in flow, computing the apparent dissociation constant of p53-MDM2 complex (130 nM, in excellent agreement with the published literature). We compared the results obtained with the ActivePipette printing and dispensing technology vs. pin spotting. Without the ActivePipette, after MDM2 addition the shift in frequency (Δf ) was 7575 Hz and the corresponding adsorbed mass was 32.9 µg. With the ActivePipette technology, after MDM2 addition Δf was 7740 Hz and the corresponding adsorbed mass was 33.6 µg. With this experiment, we confirmed the sensing potential of our device, being able to discriminate each gene and protein as well as their interactions, showing for each one of them a unique conductance curve. Moreover, we obtained a better yield with the ActivePipette technology.

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Section: Qcm_d Conductometermentioning

confidence: 99%

Section: Activepipette Piezoelectric Inkjet Technologymentioning

confidence: 99%

Section: Activepipette Piezoelectric Inkjet Technologymentioning

confidence: 99%

Section: Nappa Experimentsmentioning

confidence: 99%

Section: Nappa Experimentsmentioning

confidence: 99%

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Nanoprobe NAPPA Arrays for the Nanoconductimetric Analysis of Ultra-Low-Volume Protein Samples Using Piezoelectric Liquid Dispensing Technology

Pechkova¹,

Wiktor²,

Bragazzi³

et al. 2015

NanoWorld J

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Protein microarray generation by in situ protein expression from template DNA

Kilb

Burger

Roth

2014

Engineering in Life Sciences

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Today, whole genomes are analyzed by Next-Generation Sequencing systems, or displayed by DNA microarray in situ synthesis. However, genomic data are mainly static and do not exactly reveal the complexity of protein interaction networks of any organism or cell. To investigate protein interactions, the generation of protein microarrays, displaying whole proteomes is a prerequisite. But traditional protein microarray generation is time consuming, costly, and is restricted by a wide range of technical difficulties concerning cell culturing, protein purification, and transfer onto microarray. Some of these obstacles can be bypassed by application of cell-free expression systems, enabling fast in situ synthesis of protein microarrays without need for cell culturing. This review provides a historical timeline of the different methods to generate protein microarrays by cell-free expression, highlights differences and similarities, and reports the current state of the different approaches.

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