A sensitive three-step protocol for fluorescence-based Western blot detection

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Abstract

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.
Original languageEnglish
Pages (from-to)51-58
Number of pages8
JournalJournal of Immunological Methods
Volume334
Publication statusPublished - 2008

Keywords

  • Westernblot detection; Fluorescent conjugate; Three-stepprocedure

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