Stephen T. Yue

Quantitative Comparison of Long-wavelength Alexa Fluor Dyes to Cy Dyes: Fluorescence of the Dyes and Their Bioconjugates

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

Sensitive determination of cell number using the CyQUANT® cell proliferation assay

We describe here the development and characterization of the CyQUANT cell proliferation assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells. The assay employs CyQUANT GR dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using standard fluorescein excitation and emission wavelengths. The fluorescence emission of the dye-nucleic acid complexes correlated linearly with cell number over a large range using a wide variety of cell types. Under the recommended assay conditions, standard curves were linear (r(2)>0.995), detecting as few as 10-50 cells and as many as 25,000-50,000 cells with a single dye concentration, depending on cell type. Increasing the dye concentration extended the linear range of the assay to 100,000-250,000 cells. Results of cell proliferation and growth inhibition studies with the assay were similar to those obtained in published studies using other standard assays. CyQUANT assay measurements of serum-stimulated cell proliferation correlated well with measurements made using [3H]-thymidine. Also, the assay was used to analyze cellular DNA or RNA content, with the addition of a nuclease digestion step to the protocol. The assay procedure is simple and convenient, with no wash steps, and is readily amenable to automation.

Comparison of SYBR® Green I nucleic acid gel stain mutagenicity and ethidium bromide mutagenicity in the Salmonella/mammalian microsome reverse mutation assay (Ames test)

SYBR Green I nucleic acid gel stain is an unsymmetrical cyanine dye developed for sensitive detection of nucleic acids in electrophoretic gels. Its mechanism of nucleic acid binding is not known, whereas the most commonly used nucleic acid gel stain, ethidium bromide, is a well-characterized intercalator. We compared the mutagenicity of SYBR Green I stain with that of ethidium bromide in Salmonella/mammalian microsome reverse mutation assays (Ames tests). As expected [J. McCann, E. Choi, E. Yamasaki, B.N. Ames, Proc. Natl. Acad. Sci. USA, 72 (1975) 5135-5139], ethidium bromide showed high revertant frequencies in several frameshift indicator strains (averaging 68-fold higher than vehicle controls in TA98, 80-fold higher in TA1538, 15-fold higher in TA1537, and 4.4-fold higher in TA97a), only in the presence of rat liver extracts (S9). Small increases in revertant frequencies were observed for ethidium bromide in the base-substitution indicator strain TA102 both in the presence and absence of S9 (averaging 2.0- and 1.8-fold higher than vehicle controls, respectively) and in base-substitution indicator strain TA100 in the presence of S9 (averaging 1.6-fold higher than vehicle controls). A small mutagenic effect was detected for SYBR Green I stain in frameshift indicator strain TA98 (averaging 2. 2-fold higher than vehicle controls) only in the absence of S9 and in base-substitution indicator strain TA102, both in the presence and absence of S9 (averaging 2.2- and 2.7-fold higher than vehicle controls, respectively). Thus, SYBR Green I stain is a weak mutagen and appears to be much less mutagenic than ethidium bromide. These results suggest that SYBR Green I stain may not intercalate, and if it does, that its presence does not give rise to point mutations at a high frequency.

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate‐buffered saline or simply 150 mM NaCl using an easy, one‐step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains ‐ about 4—10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in‐gel detection of rabbit liver esterase activity using α‐naphthyl acetate and Fast Blue BB dye as well as Escherichia coli β‐glucuronidase activity using ELF‐97 β‐D‐glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.