title: A sensitive three-step protocol for fluorescence-based Western blot detection.
abstract: Western blotting is widely used in protein analysis, classically using enhanced chemoluminescence for protein detection. Fluorescence-labelled secondary antibodies have emerged in recent years for detection of antigens, in order to improve the sensitivity and the linear range of detection. Here we show that the sensitivity can be further improved by an additional step in the detection procedure: the antigen is detected by successive incubations with a primary antibody, followed by a biotinylated secondary antibody and then a tertiary fluorescent conjugate. Using the detection of different antigens by CyDye-conjugated secondary antibodies in a two-step protocol as a reference, two tertiary fluorescent conjugates were evaluated: CyDye-conjugated streptavidin and CyDye-conjugated anti-biotin antibody. An four-fold increase in sensitivity was achieved with CyDye-conjugated streptavidin; numerous unspecific bands were also generated. CyDye-conjugated anti-biotin antibody did not generate any unspecific bands and led to a 30-fold increase in sensitivity, compared to detection with CyDye-conjugated secondary antibody.
authors.affiliation: Laboratory of Biochemistry and Cell Biology (URBC), University of Namur (FUNDP), 61 rue de Bruxelles, 5000 Namur, Belgium.
authors.forename: Edouard
authors.initials: E
authors.lastname: Delaive
authors.forename: Thierry
authors.initials: T
authors.lastname: Arnould
authors.forename: Martine
authors.initials: M
authors.lastname: Raes
authors.forename: Patricia
authors.initials: P
authors.lastname: Renard
mesh_terms: D000906:Antibodies / Q000276:immunology; D001710:Biotin; D015153:Blotting, Western / Q000379:methods*; D002460:Cell Line; D005453:Fluorescence; D005456:Fluorescent Dyes; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D053533:Keratin-8 / Q000032:analysis*; D015603:Keratinocytes / Q000737:chemistry*; D016328:NF-kappa B / Q000032:analysis*; D012680:Sensitivity and Specificity; D019809:Streptavidin; D048051:p38 Mitogen-Activated Protein Kinases / Q000032:analysis*
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; C426203:KRT8 protein, human; D053533:Keratin-8; D016328:NF-kappa B; D001710:Biotin; D019809:Streptavidin; D048051:p38 Mitogen-Activated Protein Kinases