Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

• Published in: Proteomics (Weinheim) 2002-05, Vol.2 (5), p.499-512
• Main Authors: Martin, Karen, Hart, Courtenay, Schulenberg, Birte, Jones, Laurie, Patton, Wayne F.
• Format: Article
• Language: English
• Subjects: Acridines - metabolism; Alkaline Phosphatase - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western - methods; Boron Compounds - metabolism; Cattle; Dichromatic; Electroblot; Electrophoresis; Esters - metabolism; Evaluation Studies as Topic; Fluorescence; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Immunodetection; Mitochondria - chemistry; Molecular Structure; Phosphates - metabolism; Protein stain; Proteins; Proteins - analysis; Spectrometry, Fluorescence; Succinimides - metabolism
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/1615-9861(200205)2:5<499::AID-PROT499>3.0.CO;2-H

A two‐color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR‐X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39‐phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi‐ or trans‐illuminators combined with photographic or charge‐coupled device cameras, and xenon‐arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.