Secondary antibody for confocal imaging of PSD-95 was assembled in house as in (Dharmasri et al., 2023 ). Briefly, donkey anti-mouse IgG2a was mixed with NHS-Cy3B (GE PA63101) at ~13:1 molar ratio of dye:IgG for 1h at RT to achieve a final dye/IgG ratio of ~3:1, and excess dye was removed by Zeba desalting column (Thermo). Conjugated antibody was diluted to ~1.25 mg/mL in 50% glycerol, aliquoted, and stored at −20°C.
Nhs cy3b
Manufactured by GE Healthcare
NHS-Cy3B is a fluorescent dye used in various laboratory applications. It is a succinimidyl ester derivative of the cyanine dye Cy3B, which can be used to label biomolecules such as proteins, peptides, and nucleic acids. The dye exhibits excitation and emission maxima at 559 nm and 570 nm, respectively, making it suitable for detection using common fluorescence instrumentation.
Automatically generated - may contain errors
4 protocols using nhs cy3b
1
Conjugating Secondary Antibody for PSD-95 Imaging
2
Labeling Donkey Anti-Mouse IgG2a
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300 μl of donkey anti-mouse IgG2a was concentrated on a 100K MWCO Amicon spin filter (Sigma) and diluted to ~2.5 mg/mL with pH8.3 PBS, then mixed with NHS-Cy3B (GE) at ~13:1 molar ratio of dye:IgG for 1h at RT to achieve a final dye/IgG ratio of ~3:1. Excess dye was removed by Zeba 7K MWCO spin desalting column (Thermo Fisher), and the dye/protein ratio confirmed by absorbance at 559 nm and 280 nm according to the manufacturer’s instructions. Conjugated antibody was diluted to ~1.25 mg/mL in 50% glycerol, aliquoted, and stored at −20°C.
3
Oligonucleotide Labeling and Purification
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All oligonucleotides were purchased from IDT. Amino-modified oligonucleotides were labeled with Cy3B NHS (PA63101, GE Healthcare) or Atto647N NHS (18373, Sigma) in 0.1 M borate buffer (pH 8.5) and purified twice through ethanol precipitation. Please see Supporting Table 1 for complete sequence information.
4
E. coli Labeling with Fluorescent Dye
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E. coli MG1655 cells were grown overnight in LB medium and subsequently diluted 1000 times in EZ rich medium (Teknova) with 0.2% glucose. The cells were grown until the culture reached an OD600 value of 0.3 after which the cells were centrifuged at 3000 rcf for 5 minutes and resuspended in PBS buffer. The amino-reactive dye Cy3B-NHS (GE Healthcare) was added to a final concentration of 1 mM and incubated while protected from light at room temperature for 1 hour [89 (link)]. Next, the cells were centrifuged at 3000 rcf for 10 minutes and the cell pellet was resuspended in PBS.
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