Linked Life Data

17203946

Source:http://linkedlifedata.com/resource/pubmed/id/17203946

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2007-1-5
pubmed:abstractText
Plasma membrane (PM) has very important roles in cell-cell interaction and signal transduction, and it has been extensively targeted for drug design. A major prerequisite for the analysis of PM proteome is the preparation of PM with high purity. Density gradient centrifugation has been commonly employed to isolate PM, but it often occurred with contamination of internal membrane. Here we describe a method for plasma membrane purification using second antibody superparamagnetic beads that combines subcellular fractionation and immunoisolation strategies. Four methods of immunoaffinity were compared, and the variation of crude plasma membrane (CPM), superparamagnetic beads, and antibodies was studied. The optimized method and the number of CPM, beads, and antibodies suitable for proteome analysis were obtained. The PM of mouse liver was enriched 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondria was reduced 2-fold. The PM protein bands were extracted and trypsin-digested, and the resulting peptides were resolved and characterized by MALDI-TOF-TOF and ESI-Q-TOF, respectively. Mascot software was used to analyze the data against IPI-mouse protein database. Nonredundant proteins (248) were identified, of which 67% are PM or PM-related proteins. No endoplasmic reticulum (ER) or nuclear proteins were identified according to the GO annotation in the optimized method. Our protocol represents a simple, economic, and reproducible tool for the proteomic characterization of liver plasma membrane.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
1535-3893
pubmed:author
pubmed:issnType
Print
pubmed:volume
6
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
34-43
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Immunoaffinity purification of plasma membrane with secondary antibody superparamagnetic beads for proteomic analysis.
pubmed:affiliation
Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Science, Hunan Normal University, Changsha 410081, PR China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't