scholarly journals A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Brandon J. Biesiadecki ◽  
J.-P. Jin
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Protein Modification ◽  
Binding Assay ◽  
Solid Phase ◽  
Regulatory Protein ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Protein Binding Assay ◽  
Phase Protein

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

Multivalent glycosylated nanoparticles for studying carbohydrate–protein interactions

2014 ◽  
Vol 12 (30) ◽  
pp. 5563-5573 ◽  
Author(s):  
Avijit K. Adak ◽  
Hong-Jyune Lin ◽  
Chun-Cheng Lin
Keyword(s):  
Targeted Therapy ◽  
Molecular Imaging ◽  
Protein Binding ◽  
Protein Interactions ◽  
Binding Assay ◽  
Biologically Relevant ◽  
Protein Binding Assay ◽  
Multiple Copies

Glyconanoparticles decorated with multiple copies of various biologically relevant carbohydrates serve as scaffolds for protein binding assay, molecular imaging, targeted therapy, and bacterium detection.

A fully automated, continuous-flow radioimmunoassay for methotrexate.

1980 ◽  
Vol 26 (1) ◽  
pp. 97-100 ◽  
Author(s):  
R Kamel ◽  
J Landon ◽  
G C Forrest
Keyword(s):  
Electromagnetic Field ◽  
Protein Binding ◽  
Continuous Flow ◽  
Binding Assay ◽  
Solid Phase ◽  
Separation Technique ◽  
Automated Method ◽  
Protein Binding Assay ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding

Abstract We describe a fully automated continuous-flow radioimmunoassay for methotrexate. [125I]Histamine-labeled methotrexate was used as tracer. Anti-methotrexate serum was coupled to a magnetizable solid-phase and the bound and free fractions were separated with an electromagnetic field. The assay is precise (CV less than 2.5%) and rapid (30 samples per hour), incubation volume is small (about 160 micro L), and incubation brief (10 min). The accurate timing inherent in the system obviates the need to attain equilibrium, so that assay of each sample takes only 15 min. The assay is sensitive (1--100 microgram/L). There is no significant carryover between samples of high and low concentration. Results by the automated method correlated well with those by both a manual assay in which the same reagents and separation technique are used (r - 0.99) and a competitive protein-binding assay (r = 0.96).

A fully automated, continuous-flow radioimmunoassay for methotrexate.

1980 ◽  
Vol 26 (1) ◽  
pp. 97-100
Author(s):  
R Kamel ◽  
J Landon ◽  
G C Forrest
Keyword(s):  
Electromagnetic Field ◽  
Protein Binding ◽  
Continuous Flow ◽  
Binding Assay ◽  
Solid Phase ◽  
Separation Technique ◽  
Automated Method ◽  
Protein Binding Assay ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding

Abstract We describe a fully automated continuous-flow radioimmunoassay for methotrexate. [125I]Histamine-labeled methotrexate was used as tracer. Anti-methotrexate serum was coupled to a magnetizable solid-phase and the bound and free fractions were separated with an electromagnetic field. The assay is precise (CV less than 2.5%) and rapid (30 samples per hour), incubation volume is small (about 160 micro L), and incubation brief (10 min). The accurate timing inherent in the system obviates the need to attain equilibrium, so that assay of each sample takes only 15 min. The assay is sensitive (1--100 microgram/L). There is no significant carryover between samples of high and low concentration. Results by the automated method correlated well with those by both a manual assay in which the same reagents and separation technique are used (r - 0.99) and a competitive protein-binding assay (r = 0.96).

A Simplified competitive protein-binding assay for 25-hydroxycalciferol

1976 ◽  
Vol 68 (2) ◽  
pp. 99-105 ◽  
Author(s):  
B. Garcia-Pascual ◽  
A. Peytremann ◽  
B. Courvoisier ◽  
D.E.M. Lawson
Keyword(s):  
Protein Binding ◽  
Binding Assay ◽  
Protein Binding Assay ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding

24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C-18 cartridges and liquid chromatography before protein-binding assay.

1983 ◽  
Vol 29 (10) ◽  
pp. 1806-1807 ◽  
Author(s):  
M L Traba ◽  
M Babé ◽  
C de la Piedra ◽  
A Marín
Keyword(s):  
Liquid Chromatography ◽  
Protein Binding ◽  
Serum Sample ◽  
High Performance ◽  
Binding Assay ◽  
Reversed Phase ◽  
Specific Method ◽  
Dihydroxyvitamin D3 ◽  
Protein Binding Assay ◽  
24,25 Dihydroxyvitamin D3

Abstract We describe a precise, specific method for measuring 24,25-dihydroxyvitamin D3 in human serum. A 2-mL serum sample is extracted with acetonitrile and passed through a Sep-Pak C-18 cartridge. The sample is further purified by "high-performance" liquid chromatography under isocratic conditions on a normal-phase column (Radial-Pak silica-gel cartridge), then subjected to a protein-binding assay. The mean concentration of 24,25-dihydroxyvitamin D3 in serum from 22 normal adults (measured during the spring) was 2.9 micrograms/L (SD 1.9, range 6.3-0.42 microgram/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with Sep-Pak C-18 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 micrograms/L (SD 1.6 micrograms/L, n = 12), not significantly different from results with our method.

scholarly journals PathwayMatcher: proteoform-centric network construction enables fine-granularity multiomics pathway mapping

GigaScience ◽  
2019 ◽  
Vol 8 (8) ◽  
Author(s):  
Luis Francisco Hernández Sánchez ◽  
Bram Burger ◽  
Carlos Horro ◽  
Antonio Fabregat ◽  
Stefan Johansson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Knowledge Bases ◽  
Protein Isoforms ◽  
Biomedical Data ◽  
Protein Protein Interactions ◽  
Gene Sets ◽  
Functional Knowledge ◽  
Pathway Analyses ◽  
Different Levels

Abstract Background Mapping biomedical data to functional knowledge is an essential task in bioinformatics and can be achieved by querying identifiers (e.g., gene sets) in pathway knowledge bases. However, the isoform and posttranslational modification states of proteins are lost when converting input and pathways into gene-centric lists. Findings Based on the Reactome knowledge base, we built a network of protein-protein interactions accounting for the documented isoform and modification statuses of proteins. We then implemented a command line application called PathwayMatcher (github.com/PathwayAnalysisPlatform/PathwayMatcher) to query this network. PathwayMatcher supports multiple types of omics data as input and outputs the possibly affected biochemical reactions, subnetworks, and pathways. Conclusions PathwayMatcher enables refining the network representation of pathways by including proteoforms defined as protein isoforms with posttranslational modifications. The specificity of pathway analyses is hence adapted to different levels of granularity, and it becomes possible to distinguish interactions between different forms of the same protein.

A Competitive Protein-Binding Assay for 25-Hydroxycholecalciferol

1975 ◽  
Vol 17 (2) ◽  
pp. 57-57
Author(s):  
Yoshiki Seino ◽  
Tsunesuke Shimotsuji ◽  
Shintaro Okada ◽  
Teisuke Hiejima ◽  
Chiiko Ikehara ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Assay ◽  
Protein Binding Assay ◽  
Competitive Protein Binding Assay ◽  
Competitive Protein Binding ◽  
25 Hydroxycholecalciferol
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