Linked Life Data

19834902

Source:http://linkedlifedata.com/resource/pubmed/id/19834902

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
24
pubmed:dateCreated
2009-12-22
pubmed:abstractText
The comprehensive and quantitative analysis of the protein phosphorylation patterns in different cellular context is of considerable and general interest. The ability to quantify phosphorylation of discrete signalling proteins in large collections of biological samples would greatly favour the development of systems biology in the field of cell signalling. Reverse-phase protein array (RPPA) potentially represents a very attractive approach to map signal transduction networks with high throughput. In the present report, we describe an improved detection method for RPPA combining near-infrared with one or two rounds of tyramide-based signal amplification. The LOQ was lowered from 6.84 attomoles with a direct detection protocol to 0.21 attomole with two amplification steps. We validated this method in the context of intracellular signal transduction triggered by follicle-stimulating hormone in HEK293 cells. We consistently detected phosphorylated proteins in the sub-attomole range from less than 1 ng of total cell extracts. Importantly, the method correlated with Western blot analysis of the same samples while displaying excellent intra- and inter-slide reproducibility. We conclude that RPPA combined with amplified near-infrared detection can be used to capture the subtle regulations intrinsic to signalling network dynamics at an unprecedented throughput, from minute amounts of biological samples.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1615-9861
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
9
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
5446-54
pubmed:meshHeading
pubmed:year
2009
pubmed:articleTitle
A highly sensitive near-infrared fluorescent detection method to analyze signalling pathways by reverse-phase protein array.
pubmed:affiliation
BIOS group, INRA, Unité Physiologie de la Reproduction et des Comportements, Nouzilly, France.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't, Validation Studies