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Alexa fluor 488 c5 maleimide af 488 mal

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Alexa Fluor™ 488 C5 Maleimide (AF-488-Mal) is a fluorescent labeling reagent. It is a derivative of the Alexa Fluor™ 488 dye conjugated to a maleimide functional group.

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Alexa Fluor 488 C5 maleimide Alexa 488 C5-maleimide Alexa 488 maleimide Alexa Fluor 488 AF 488 Alexa 488 Alexa Fluors

3 protocols using alexa fluor 488 c5 maleimide af 488 mal

1

Fluorescent Labeling of Bacterial Pili

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Alexa Fluor™ 488 C5 Maleimide (AF-488-Mal; Thermo Fisher Scientific; Cat# A10254) was dissolved in DMSO, aliquoted and stored at -20°C protected from light. Cultures used for staining were grown for 3.5h at 30°C on the rotary wheel, as above, in the absence and presence of competence induction, as required. To stain cells 100 µL of culture was mixed with dye at a final concentration of 25 µg/mL33 (link) and incubated at RT for 5 min in the dark. Stained cells were harvested by centrifugation (5000 x g; 1 min), washed once with LB, re-suspended in 200 µL LB and imaged immediately. For quantification of piliation in snapshot imaging approximately 2000 cells per strain were analysed in each of three independent repeats. A subset of this data set was also analysed for the number of pili per cell and pilus length, as indicated in the text. For time-lapse analysis, images were acquired at RT at 10 sec intervals for 1 min (i.e. 7 frames). To analyse the number of pili produced per cell, per min, 5 fields of cells were analysed for each of the three independent repeats, yielding an analysis of 1947 cells in total.
Adams D.W., Stutzmann S., Stoudmann C, & Blokesch M. (2019). DNA-uptake pili of Vibrio cholerae are required for chitin colonisation and capable of kin recognition via sequence-specific self-interaction. Nature microbiology, 4(9), 1545-1557.
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2

Fluorescent Labeling of Bacterial Pili

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Alexa Fluor™ 488 C5 Maleimide (AF-488-Mal; Thermo Fisher Scientific; Cat# A10254) was dissolved in DMSO, aliquoted and stored at -20°C protected from light. Cultures used for staining were grown for 3.5h at 30°C on the rotary wheel, as above, in the absence and presence of competence induction, as required. To stain cells 100 µL of culture was mixed with dye at a final concentration of 25 µg/mL33 (link) and incubated at RT for 5 min in the dark. Stained cells were harvested by centrifugation (5000 x g; 1 min), washed once with LB, re-suspended in 200 µL LB and imaged immediately. For quantification of piliation in snapshot imaging approximately 2000 cells per strain were analysed in each of three independent repeats. A subset of this data set was also analysed for the number of pili per cell and pilus length, as indicated in the text. For time-lapse analysis, images were acquired at RT at 10 sec intervals for 1 min (i.e. 7 frames). To analyse the number of pili produced per cell, per min, 5 fields of cells were analysed for each of the three independent repeats, yielding an analysis of 1947 cells in total.
Adams D.W., Stutzmann S., Stoudmann C, & Blokesch M. (2019). DNA-uptake pili of Vibrio cholerae are required for chitin colonisation and capable of kin recognition via sequence-specific self-interaction. Nature microbiology, 4(9), 1545-1557.
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3

Site-Specific Pilus Labeling Protocol

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Pilus labeling was performed as previously described with minor modifications (23 (link), 50 (link)). Briefly, control strains or genetically modified bacteria with modified pilA alleles (encoding site-specific amino acid changes to knock-in a new cysteine residue) were pregrown overnight at 37°C, back diluted 1:100 in 2 ml LB (supplemented without or with l-arabinose, as indicated), and grown at 37°C until they reached an OD600 of approximately 0.65. Alexa Fluor 488 C5 maleimide (AF-488-Mal; Thermo Fisher Scientific) was added to 100 μl of the bacterial culture at a final concentration of 25 μg/ml, gently mixed, and incubated for 15 min at room temperature in the dark. Cells were subsequently harvested by centrifugation (5,000 × g for 1 min.), washed once in 1× PBS, and resuspended in 30 μl of PBS before being imaged as described above.
Vesel N, & Blokesch M. (2021). Pilus Production in Acinetobacter baumannii Is Growth Phase Dependent and Essential for Natural Transformation. Journal of Bacteriology, 203(8), e00034-21.
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