Labeling Proteins with Fluorescent Dye

AtTCP21 and melittin were labeled by adding Flamma^® 675-NHS (BioActs, Incheon, Republic of Korea), a fluorescent dye solution, to the proteins in buffer A at a molar ratio of 1:1. After 2 h of incubation, the mixtures were dialyzed against PBS for 48 h to remove any unbound fluorescent dye. The labeled protein samples were incubated with C. gloeosporioides cells at 28 °C for 4 h. The fungal cells were washed thrice with buffer A, mounted on a cover glass in 50% glycerol and 0.1% n-propyl gallate solution, and examined under a CLSM (A1R HD 25, Nikon, Japan) [19 (link)].

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Park S.C., Yoon A.M., Kim Y.M., Lee M.Y, & Lee J.R. (2023). Antifungal Action of Arabidopsis thaliana TCP21 via Induction of Oxidative Stress and Apoptosis. Antioxidants, 12(9), 1767.

Publication 2023

A1R HD 25

Buffer Cells Fluorescent dye Glycerol Melittin Molar Propyl gallate Protein Proteins a

Corresponding Organization : Texas A&M University

Other organizations : Sunchon National University, Jeonbuk National University, Daejeon Health Institute of Technology

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Variable analysis

independent variables

Labeling of AtTCP21 and melittin with Flamma® 675-NHS (a fluorescent dye)

dependent variables

Binding of labeled AtTCP21 and melittin to C. gloeosporioides cells

control variables

Buffer A used for labeling proteins
Incubation time of 2 h for labeling
Dialysis against PBS for 48 h to remove unbound dye
Incubation of labeled proteins with C. gloeosporioides cells at 28 °C for 4 h
Washing of fungal cells thrice with buffer A
Mounting of cells on cover glass in 50% glycerol and 0.1% n-propyl gallate solution
Examination under a CLSM (A1R HD 25, Nikon, Japan)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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