Use of protein–acrylamide copolymer hydrogels for measuring protein concentration and activity

Brueggemeier, Shawn B.; Kron, Stephen J.; Palecek, Sean P.

doi:10.1016/j.ab.2004.02.012

Cited by 26 publications

(27 citation statements)

References 26 publications

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“…After attachment of the proteinacrylamide copolymer spots, the slides were washed as above in TBST and subsequently imaged on an inverted epi fluorescence Olympus microscope (Olympus, Melville, NY). 36 Array-Based Kinase Assays. Just prior to the kinase assay, the glass slides were removed from the Abl kinase assay buffer and dried under a stream of compressed air.…”

Section: Methodsmentioning

confidence: 99%

“…Also, the covalent nature of the protein-acrylamide copolymerization strategy ensures stable immobilization of proteins, even over extended periods of protein array storage. 36 The porous, three-dimensional shape of our proteinacrylamide copolymer array spots provides several advantages over traditional, two-dimensional protein immobilization techniques. The capacity of our hemispherical spot is greatly increased compared to strategies in which proteins are directly attached to the solid surface.…”

Section: Protein Array-based Detection Ofmentioning

confidence: 99%

“…Here we have detected up to 40 GSTCrkl spots per microscope slide; however, with commercially available microarray printing techniques densities as high as thousands of spots per slide are possible. 36 PCR-based methods are also capable of diagnosing CML at the molecular level by the presence of the BCR-ABL fusion gene or BCR-ABL mRNA transcript. Quantitative RT-PCR can detect residual disease at levels as low as one leukemic cell in 10 5 -10 6 , and increased levels of BCRABLexpressionhavebeenassociatedwithdiseaseprogression.…”

Section: Protein Array-based Detection Ofmentioning

confidence: 99%

“…36 Clean glass slides are treated with (3-acryloxpropyl)trimethoxysilane, forming an acrylic-functionalized surface. Free acrylamide monomer and the cross-linker Bis are then polymerized to the acrylic-functionalized surface, resulting in a polyacrylamide-coated surface (Scheme 1A).…”

Section: Fabrication Of Gst-crkl Arraysmentioning

confidence: 99%

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Protein−Acrylamide Copolymer Hydrogels for Array-Based Detection of Tyrosine Kinase Activity from Cell Lysates

Brueggemeier

Kron

et al. 2005

Biomacromolecules

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We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylic-functionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 microM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied.

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Section: Methodsmentioning

confidence: 99%

Section: Protein Array-based Detection Ofmentioning

confidence: 99%

Section: Protein Array-based Detection Ofmentioning

confidence: 99%

Section: Fabrication Of Gst-crkl Arraysmentioning

confidence: 99%

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Protein−Acrylamide Copolymer Hydrogels for Array-Based Detection of Tyrosine Kinase Activity from Cell Lysates

Brueggemeier

Kron

et al. 2005

Biomacromolecules

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“…52 Enzyme-kinetic study has been performed in microfluidic formats. [53][54][55][56][57][58] Important to proteomics, enzymatic digestion before MALDI-TOF/MS (Matrix-assisted laser desorption-ionization time-of-fly/mass spectrometry) peptide mapping of a protein has been explored in microfluidic devices. [59][60][61][62][63][64][65][66] Compared to conventional in-solution enzyme digestion, which is time consuming and offers limited sensitivity, microfluidic formats have shown high conversion rates, facile replacement of inactivated enzyme, and long-term stability.…”

Section: Introductionmentioning

confidence: 99%

Protein immobilization techniques for microfluidic assays

2013

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Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization.

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Use of High‐Throughput Proteomic Arrays for the Discovery of Disease‐Associated Molecules

Molina

Morrow

Liang

2010

Pharmaceutical Sciences Encyclopedia

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The high‐throughput proteomics approach is the natural progression of the genome sequencing projects of the 1990s. Organisms such as M. tuberculosis with 4000 ORFs, or malaria ( P. falciparum and P. vivax ) with even larger genomes, can now be assayed on a whole‐proteome scale. This article focuses on assay miniaturization technology and applications of advances in cloning and gene expression in profiling the immune response to infection, cancer, and autoimmune disease.

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Use of protein–acrylamide copolymer hydrogels for measuring protein concentration and activity

Cited by 26 publications

References 26 publications

Protein−Acrylamide Copolymer Hydrogels for Array-Based Detection of Tyrosine Kinase Activity from Cell Lysates

Protein−Acrylamide Copolymer Hydrogels for Array-Based Detection of Tyrosine Kinase Activity from Cell Lysates

Protein immobilization techniques for microfluidic assays

Use of High‐Throughput Proteomic Arrays for the Discovery of Disease‐Associated Molecules

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