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4-Color Imaging with DyLight 405 Conjugates

DyLight fluorescent dyes are a new family of dyes with improved brightness and photostability. The new DyLight 405-conjugated secondary antibodies are excited maximally at about 400 nm and fluoresce with a peak at about 421 nm (Figure 1). They are very bright and photostable when used with confocal microscopes equipped with a 405 nm laser and appropriate emission filter (Figure 2).

Under these conditions, it is possible to perform effective 4-color imaging with good color separation, good photostability, and high sensitivity in both aqueous and permanent mounting media. The combination of DyLight 405, DyLight 488, Rhodamine Red-X, and DyLight 649 provides for maximum color separation (Figure 3).

Other 4-color dye combinations, which may be equally effective but have slightly less color separation, include DyLight 405, DyLight 488, DyLight 549 (or Cy3), and DyLight 649 (Figure 4).

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Peak wavelengths of absorption and emission maxima for DyLight-conjugated, affinity-purified secondary antibodies from Jackson ImmunoResearch.
  Figure 2. Mouse retinal tissue stained with DyLight 405 (pseudo-colored green)-donkey anti-rabbit IgG (JIR) with rabbit anti-GFAP (DAKO), DyLight 488 (pseudo-colored red) -donkey
Figure 1. Excitation (violet) and emission (blue) spectra for DyLight 405 conjugated to an affinity-purified secondary antibody from Jackson ImmunoResearch.   anti-mouse IgG (JIR) with mouse anti-neurofilament (Abcam), and DyLight 649 (pseudo-colored blue)-donkey anti-goat IgG (JIR) with goat anti-collagen IV (Chemicon).
  Figure 4. Mouse retinal tissue stained with DyLight 405 (pseudo-colored green)-donkey anti-rabbit IgG (JIR) with rabbit anti-GFAP (DAKO),DyLight 488 (pseudo-colored red)
Figure 3. Emission spectra of DyLight 405 (blue), DyLight 488 (green), Rhodamine Red-X (red), and DyLight 649 (brown) following conjugation to antibodies. The figure shows that effective 4-color imaging can be performed with maximum color separation using these dyes.   -donkey anti-mouse IgG (JIR) with mouse anti-neurofilament (Abcam), Cy3 (pseudo-colored blue)-donkey anti-chicken IgG (JIR) with chicken anti-vimentin (Chemicon), and DyLight 649 (pseudo-colored white)- donkey anti-goat IgG (JIR) with goat anti-collagen IV (Chemicon).

Anti-Goat IgG, Light Chain Specific or Western Blotting after immunoprecipitation

Monoclonal Mouse Anti-Goat IgG, Light Chain Specific is a new addition to our family of anti-Light Chain specific antibodies for Western blotting after immunoprecipitation (IP) of antigens with primary antibodies. Anti-Light Chain specific antibodies react with native primary antibodies used for detecting specific protein bands on Western blots (Figure A). If diluted properly, anti-Light Chain specific antibodies do not bind to the reduced and denatured IgG heavy chain band (50kDa) on blots (Figure B,D, E, and F). Therefore by using anti-Light Chain specific antibodies , detection of antigens with molecular weights near 50 kDa is not obscured by large amounts of reduced and denatured IgG heavy chains from primary antibodies used for immunoprecipitation. View a complete listing of anti-light chain antibody conjugates

Detection of primary antibodies made in goat (against a 50kDa protein) by mouse anti-goat IgG, Light Chain (LC) specific (A). Heavy (50kDa) and light (25kDa) chains of reduced and SDS-denatured mouse IgG (B andC), goat IgG (D), rat IgG (E), and rabbit IgG (F) were separated by SDS-PAGE and detected on Western blots using HRP-goat anti-mouse IgG, LC specific (B), HRP-goat anti-mouse IgG (H+L) (C), HRP-mouse anti-goat IgG, LC specific (D), HRP-goat anti-rat IgG, LC specific (E), and HRP-mouse anti-rabbit IgG, LC specific (F), respectively. No heavy chain band was detected when anti-IgG, LC specific antibodies were used for detection (B, D, E, and F). However, both heavy and light chain bands were detected with anti-IgG (H+L) (C).
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