Detection of Texas red-labelled double-stranded DNA by non-enzymatic peroxyoxalate chemiluminescence.

We have found previously that different fluorescent dyes cannot be efficiently excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H(2)O(2) reaction when they are intercalated between the DNA bases or bound to the minor groove of the double helix. Here we show that the fluorescent dye Texas red, covalently bound to the 3' ends of double-stranded DNA molecules, exhibits a high emission intensity when excited by the TCPO-H(2)O(2) reaction. In this case, the charge transfer between the intermediate produced in the peroxyoxalate chemiluminescent reaction and Texas red can take place because this fluorophore is not buried inside the DNA structure. We describe the application of this chemiluminescent reaction to the detection of blotted DNA on nylon membranes.

Green-light transilluminator for the detection without photodamage of proteins and DNA labeled with different fluorescent dyes.

The excitation spectra of Nile red and SYPRO red, two currently used dyes for the fluorescent staining of protein bands in sodium dodecyl sulfate (SDS)-polyacrylamide gels, show an excitation peak in the UV region and another in the visible region (maximum at about 550 nm). Ethidium bromide and other intercalating dyes, e.g. propidium iodide, ethidium dimers, and benzoxazolium-4-quinolinium dimer-3 (YOYO), used for the fluorescent staining of DNA bands in agarose gels also show an excitation peak in the same region of the visible spectrum. We have designed and constructed a green-light transilluminator with an emission maximum at 542 nm. This visible transilluminator allows the detection of protein bands stained with Nile red and SYPRO red with the same sensitivity obtained with a 300 nm UV transilluminator. The green-light transilluminator also allows the detection of about 2 ng of DNA per band in gels stained with ethidium bromide and the other intercalating dyes indicated above. In contrast to the UV transilluminators, the green-light transilluminator does not produce photodamage of DNA even after long exposures (10 min). This makes this transilluminator very useful for preparative work. Furthermore, the green-light transilluminator does not require UV safety equipment and, consequently, it can be very convenient for teaching laboratories.

Inhibition of peroxyoxalate chemiluminescence by intercalation of fluorescent acceptors between DNA bases.

We have examined the ability of different fluorescent DNA dyes to become chemically excited by the peroxyoxalate chemiluminescent reaction. The intercalating dyes ethidium bromide and propidium iodide, and the bis-intercalating dyes ethidium homodimer-1, benzoxazolium-4-pyridinium dimer-1 and benzoxazolium-4-quinolinium dimer-1, exhibit an intense chemiluminescence when they are excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction in the absence of DNA. However, the chemiluminescence of these dyes is very low when they are bound to double-stranded DNA (dsDNA). In contrast, the minor groove-binding dye Hoechst 33258 excited by the TCPO-H2O2 reaction shows approximately the same chemiluminescence intensity when it is free in solution or complexed with dsDNA. Structural alterations or partial dissociation of dsDNA-bis-intercalating dye complexes produced by the addition of acetone, NaCl, MgCl2 or the cationic surfactant cetyltrimethylammonium bromide increases the chemiluminescence intensity. A moderate chemiluminescence intensity is observed when bis-intercalating dyes are complexed with single-stranded DNA. Our results indicate that the energy from the intermediates produced in the peroxyoxalate chemiluminescent reaction cannot be efficiently transferred to fluorescent dyes complexed with DNA; chemiexcitation is almost completely inhibited when dyes are buried in the dsDNA structure by intercalation between the base pairs.

Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate-polyacrylamide gels and western blots before immunodetection and sequencing.

The fluorogenic dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge-coupled device camera. Electrophoretic bands containing 5-10 ng of protein can be detected on the MDPF-stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate-polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625-626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N-terminal microsequencing and immunodetection of specific bands with polyclonal antibodies.

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.