Immunoprecipitation of Proteins under Nondenaturing Conditions

TY - CHAP

T1 - Immunoprecipitation of Proteins under Nondenaturing Conditions

AU - Lukas, Jiri

AU - Bartek, Jiri

AU - Hansen, Klaus

PY - 2006/12/1

Y1 - 2006/12/1

N2 - Immunoprecipitation of native proteins has proven to be a powerful and widely used approach in addressing questions related to the nature of a single protein or protein complexes existing under different biological conditions. Combined with the recent improvements of protein microsequencing techniques and mass spectrometry, immunoprecipitation also gives the researcher an option to obtain sequence information from unknown proteins identified through coimmnunoprecipitation and thereby collect data on multiprotein complexes. A critical prerequisite for successful analysis of immunoprecipitated native proteins is the quality of the primary antigen-specific antibodies. For the most straightforward interpretation of results, such a reagent should form specific immunocomplexes with the antigen in its native form without dissociating other associated proteins. In several cases it can be advantageous to avoid the scraping of cells into PBS and to perform a more instant lyses procedure based on adding lyses buffer directly to the cell monolayer that has been washed previously three times with ice-cold phosphate-buffered saline (PBS). © 2006

AB - Immunoprecipitation of native proteins has proven to be a powerful and widely used approach in addressing questions related to the nature of a single protein or protein complexes existing under different biological conditions. Combined with the recent improvements of protein microsequencing techniques and mass spectrometry, immunoprecipitation also gives the researcher an option to obtain sequence information from unknown proteins identified through coimmnunoprecipitation and thereby collect data on multiprotein complexes. A critical prerequisite for successful analysis of immunoprecipitated native proteins is the quality of the primary antigen-specific antibodies. For the most straightforward interpretation of results, such a reagent should form specific immunocomplexes with the antigen in its native form without dissociating other associated proteins. In several cases it can be advantageous to avoid the scraping of cells into PBS and to perform a more instant lyses procedure based on adding lyses buffer directly to the cell monolayer that has been washed previously three times with ice-cold phosphate-buffered saline (PBS). © 2006

UR - http://www.scopus.com/inward/record.url?scp=84884878339&partnerID=8YFLogxK

U2 - 10.1016/B978-012164730-8/50216-1

DO - 10.1016/B978-012164730-8/50216-1

M3 - Book chapter

AN - SCOPUS:84884878339

SN - 9780121647308

VL - 4

SP - 253

EP - 258

BT - Cell Biology, Four-Volume Set

PB - Elsevier Science Inc.

ER -