Development of chemiluminescent and fluorescent methods for rotein and dna detection
- Autores: Francisco Javier Alba González
- Directores de la Tesis: Juan-Ramón Dabán Balañá (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2001
- Idioma: español
- Tribunal Calificador de la Tesis: Jordi Bartroli Molins (presid.), Jaume Farrés Vicén (secret.), Xavier Fernández Busquets (voc.)
- Materias:
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- Resumen
This doctoral thesis deals with: (1) the development of fluorescent detection procedures and strategies for the sensitive visualization of proteins in gels and blots, and DNA in agarose gels, and (2) the use of the peroxyoxalate chemiluminescence to develop new detection methods for proteins and DNA.
We have carried out a detailed study to reduce the background fluorescence of gels stained with the original Nile red procedure developed in our group. A lower Nile red concentration in water (2 ug/ml) gave the best results among the different staining protocols assayed. The low background after a 5-minute staining allows the detection of 5 ng of protein per band. In addition, the excitation spectra of currently used dyes for the fluorescent staining of proteins (including Nile red) and DNA in electrophoretic gels show an excitation peak in the visible region (about 550 nm). We have designed a green-light transilluminator (emission maximum at 542 nm) that allows the detection of protein and DNA bands in the low nanogram level. In contrast to the ultraviolet (UV) transilluminators, the green-light transilluminator does not produce photodamage of DNA even after long exposures (10 min), making it very useful for preparative work. Furthermore, it can be very convenient for teaching laboratories because it does not require UV safety equipment.
We have found that proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) can be chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. MDPF-labeled proteins can be detected in solution and on PVDF membranes. The method we have developed allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Alternatively, MDPF-labeled proteins can be visualized by transillumination with UV light with a detection limit of 0.5 ng of protein on slot blot and 5-10 ng on Western blot. Previous visualization of gels with Nile red does not interfere with MDPF staining and fluorescent detection of the Western blots thus allowing monitoring of total protein patterns after electrophoresis and blotting. Neither the chemiluminescent nor the fluorescent detection preclude further microsequencing and immunodetection of specific bands.
Our results show that different intercalating and bis-intercalating fluorescent DNA dyes exhibit an intense chemiluminescence when they are excited by the TCPO-H2O2 reaction in absence of DNA. However, their chemiluminescence is very low when they are bound to double-stranded DNA (dsDNA). In contrast, the minor groove-binding dye Hoechst 33258 shows approximately the same chemiluminescence intensity when it is free in solution or bound to dsDNA. Partial dissociation of dsDNA-bis-intercalating dye complexes produced by the addition of acetone, NaCl, MgCl2 or CTAB increases the chemiluminescence and a moderate emission is observed when bis-intercalating dyes are complexed with single-stranded DNA. These results indicate that the energy from the intermediates produced in the peroxyoxalate chemiluminescent reaction cannot be efficiently transferred to fluorescent dyes buried in the dsDNA structure by intercalation between the base pairs. However, Texas red covalently bound to the 3 ends of dsDNA molecules exhibits a high emission intensity when excited by the reaction. In this case, the energy produced in the TCPO-H2O2 reaction can be transferred to the Texas red because it is not buried inside the DNA structure. This chemiluminescence can be applied to the detection of DNA blotted onto nylon membranes.
