Peptides were captured on PCA resin as described. After rinsing, the C-terminus was first coupled with 100 mM propargylamine, 100 mM HCTU, and 100 mM DIEA in DMF for 2 hours at room temperature. The resin was extensively washed with DMF and the Lys residues were labelled with 0.5 mM Atto647N-NHS (Attotec) and 1 mM DIEA overnight at room temperature. The resin was washed extensively in DMF and DCM and all of the peptides cleaved from the resin with a TFA cocktail (95 % TFA, 2.5 % H2O, and 2.5 % TIS) for 2.5 hours. The supernatant was collected and concentrated with a N2 stream. Ice cold diethyl ether is added (10 vol) and the peptides collected by centrifugation for 10 minutes at 17,000 g. The peptide was analysed by high-resolution mass spectrometry to confirm the double labelling.
Atto647n nhs
Manufactured by ATTO-TEC
Atto647N-NHS is a fluorescent dye molecule developed by ATTO-TEC for labeling proteins and other biomolecules. It has an excitation maximum at 646 nm and an emission maximum at 662 nm, making it suitable for detection in the red region of the visible spectrum.
Automatically generated - may contain errors
4 protocols using atto647n nhs
1
Peptide Labeling and Purification
2
Fluorescently Labeled RNA Protocol
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For fluorophone labeling, 10 nmol of each RNA were dissolved in 150 µL borate-buffer (0.1 M sodium tetraborate (Merck), pH 8.4). GFP_1, PSD95_1, CAMK2_1 and β-actin_1 were incubated with 200 nmol ATTO565 NHS (ATTO-TEC), dissolved in 50 µL DMF (Lumiprobe, labeling grade), for 4 h at 37 °C. PSD95_2, CAMK2_2 and Beta Actin_2 were incubated with 200 nmol ATTO647N NHS (ATTO-TEC), dissolved in 50 µL DMF, for 4 h at 37 °C. Buffer and the excess of fluorophore were removed by size-exclusion chromatography (NAP 25, GE Healthcare). The solvent was evaporated at 4 °C using a vacuum concentrator. The residue was purified by RP-HPLC on an Agilent 1200 equipped with an Xbridge BEH C18 OBD (300 Å, 3.5 µm, 4.6 × 250 mm, 1 mL/min, 60 °C). As solvents 400 mM hexafluoroisopropanol, 16.3 mM Et3N, pH 8.3 and MeOH were used with a gradient from 5% MeOH to 100% MeOH in 50 min.
3
In Vivo Staining and Clearing of Lymph Nodes
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Terminally anesthetized Ccl19-cre hem; MADM-7 GT/TG mice (mix C57BL/6J and CD1 background) were in vivo stained by retro-orbital injection of 40 µg/PBS mouse α-peripheral node addressin (PNAd) concentrated hybridoma supernatant labeled with Atto-647N-NHS (Atto-Tec). After 10 min, popliteal LNs were harvested and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) overnight at 4°C. Samples were washed in PBS, cleaned under a stereomicroscope and cleared with the CUBIC protocol 52 . Briefly, samples were incubated in CUBIC reagent 1 for 3 days at 37°C, which was replaced every 24h. Samples were then washed with PBS, embedded in 2% low-meting temperature agarose (Sigma-Aldrich), and sequentially dehydrated in 30% sucrose (Sigma-Aldrich) (1 day at 4°C) and 50% sucrose (2 days at 4°C). Finally, samples were incubated in CUBIC reagent 2 for 2 days at RT. Cleared samples were imaged using a custom LSFM setup 53 . Acquired images were stitched using the FIJI Grid/Collection-stitching plugin (Preibitsch Laboratory), despeckled and manually registered using a custom-alignment-tool in Matlab (developed by Ekaterina Papusheva).
4
Fluorescent Lipid Synthesis and Analysis
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AlexaFluor647 and DiI were obtained from Invitrogen; Atto647N-NHS was from Attotec. Cholesterol linked to boron dipyrromethene difluoride at sterol carbon-24 (C-BodipyFL) was synthesized as described [27] and Cholesteryl ester-BodipyFL (CE-BodipyFL) was synthesized by esterifying linoleic acid with C-BodipyFL [28] .
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