TY - JOUR
T1 - A sensitive three-step protocol for fluorescence-based Western blot detection
AU - Delaive, Edouard
AU - Arnould, Thierry
AU - Raes, Martine
AU - Renard, Patricia
N1 - Publication code : RES. ACAD.
PY - 2008
Y1 - 2008
N2 - Westernblottingis widelyusedinproteinanalysis, classicallyusingenhancedchemoluminescencefor proteindetection.
Fluorescence-labelledsecondaryantibodies haveemergedinrecent years for detectionof antigens, inorder toimprovethe
sensitivityandthelinear rangeof detection. Hereweshowthat thesensitivitycanbefurther improvedbyanadditional step
inthedetectionprocedure:theantigenisdetectedbysuccessiveincubationswithaprimaryantibody, followedbyabiotinylated
secondaryantibodyandthenatertiaryfluorescent conjugate. Usingthedetectionof different antigensbyCyDye-conjugated
secondary antibodies in a two-step protocol as a reference, two tertiary fluorescent conjugates were evaluated: CyDye-
conjugatedstreptavidinandCyDye-conjugatedanti-biotinantibody. Anfour-foldincrease insensitivitywas achievedwith
CyDye-conjugatedstreptavidin;numerousunspecificbandswerealsogenerated. CyDye-conjugatedanti-biotinantibodydidnot
generate anyunspecific bands andledtoa 30-foldincrease insensitivity, comparedtodetectionwithCyDye-conjugated
secondaryantibody.
©2008Elsevier B.V. All rightsreserved.
AB - Westernblottingis widelyusedinproteinanalysis, classicallyusingenhancedchemoluminescencefor proteindetection.
Fluorescence-labelledsecondaryantibodies haveemergedinrecent years for detectionof antigens, inorder toimprovethe
sensitivityandthelinear rangeof detection. Hereweshowthat thesensitivitycanbefurther improvedbyanadditional step
inthedetectionprocedure:theantigenisdetectedbysuccessiveincubationswithaprimaryantibody, followedbyabiotinylated
secondaryantibodyandthenatertiaryfluorescent conjugate. Usingthedetectionof different antigensbyCyDye-conjugated
secondary antibodies in a two-step protocol as a reference, two tertiary fluorescent conjugates were evaluated: CyDye-
conjugatedstreptavidinandCyDye-conjugatedanti-biotinantibody. Anfour-foldincrease insensitivitywas achievedwith
CyDye-conjugatedstreptavidin;numerousunspecificbandswerealsogenerated. CyDye-conjugatedanti-biotinantibodydidnot
generate anyunspecific bands andledtoa 30-foldincrease insensitivity, comparedtodetectionwithCyDye-conjugated
secondaryantibody.
©2008Elsevier B.V. All rightsreserved.
KW - Westernblot detection; Fluorescent conjugate; Three-stepprocedure
UR - http://www.scopus.com/inward/record.url?scp=41949097627&partnerID=8YFLogxK
M3 - Article
VL - 334
SP - 51
EP - 58
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
ER -