Fluorescent labeling of proteins with Nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: Physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots

Daban, Joan‐Ramon

doi:10.1002/1522-2683()22:5<874::aid-elps874>3.0.co;2-u

Cited by 26 publications

(6 citation statements)

References 34 publications

(74 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Deletion of efflux pump genes affects drug accumulation. Rhodamine 6G and Nile Red are fluorescent dyes which have been used to determine the function of efflux pumps (34)(35)(36)(37)(38). It is possible that modification of the genomic constituents of the efflux pumps could alter the levels of accumulation of the fluorescent dyes and reflect the function of the efflux pumps.…”

Section: Resultsmentioning

confidence: 99%

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment

Chang

Sionov

Lamichhane

et al. 2018

Antimicrob Agents Chemother

View full text Add to dashboard Cite

and species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, H99 and R265. Deletion of in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when or was deleted in both strains. However, when these genes were deleted in combination with , a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in mutants of both strains, but rhodamine 6G accumulation increased only in the mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. and expression was upregulated in H99 and R265 when treated with fluconazole. However, expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

show abstract

Section: Resultsmentioning

confidence: 99%

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment

Chang

Sionov

Lamichhane

et al. 2018

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…Nile Red can be employed as sensitive staining approach, detecting about 5 ng protein per band. As compared to other staining protocols, Nile Red staining has the advantage of being a fast one-step procedure ( 127 ). Alternatively, the dyes Sypro Orange (excitation maximum 472 nm, emission maximum 569 nm) and Sypro Red (excitation maximum 547 nm, emission maximum 635 nm) can be used for staining of SDS-PAGE gels and Western blots with a sensitivity of 1 to 10 ng protein per band.…”

Section: Applications Of Fluorescent Dyes In Protein Characterizationmentioning

confidence: 99%

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

2008

View full text Add to dashboard Cite

Abstract. Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

show abstract

“…Nile Red is a fluorescent dye, known as a probe of intracellular lipids and hydrophobic domains of proteins [25]. It is a vital, lipid-soluble, highly selective stain for yeast lipid particles [26].…”

Section: Introductionmentioning

confidence: 99%

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Ivnitski-Steele

Holmes

Lamping

et al. 2009

Analytical Biochemistry

108

View full text Add to dashboard Cite

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter, CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile Red as a new fluorescent substrate for CaCdr1p, CaCdr2p and CaMdr1p. Nile Red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile Red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile Red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.

show abstract

Fluorescent labeling of proteins with Nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: Physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots

Cited by 26 publications

References 34 publications

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment

Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Contact Info

Product

Resources

About