Full-length proteins were expressed in HEK293 mammalian expression system. To test LPHN3 WT and mutants expression, transfected cells were stained with mouse anti-FLAG M2 antibodies, 1:1000 (F3165, “Sigma”). Fluorescence was determined by incubating with anti-mouse FITC. To test FLRT3 WT and mutant expression, cells were stained with mouse anti c-Myc antibodies (9E10, “DSHB”) 1:20 and anti-mouse FITC, 1:100. For binding assays purified monomeric or tetrameric soluble proteins were added to primary antibodies. Precomplex of BTtrisNTA - NeutrAvidinDyLight 650 (NAV650) (84607, “Thermo Scientific”) or only NeutrAvidinDyLight 650 (NAV650) was used for fluorescent labeling.
Neutravidindylight 650
Manufactured by Thermo Fisher Scientific
NeutrAvidinDyLight 650 is a biotin-binding protein conjugated with the DyLight 650 fluorescent dye. It is used for the detection and quantification of biotinylated proteins, peptides, or other biomolecules in various assays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant rnasin ribonuclease inhibitor, by Promega (1 mentions)
Cd14 fitc, by Immunotools (1 mentions)
Facsaria sorp machine, by BD (1 mentions)
96 well pcr plate, by Eppendorf (1 mentions)
Cd19 bv605, by BD (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Cd27 pecf594, by BD (1 mentions)
Cd3 fitc, by Immunotools (1 mentions)
3 protocols using neutravidindylight 650
1
Quantifying Protein Expression and Binding
2
Biotinylated Microsphere Functionalization with NeutrAvidin Dye
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The biotinylated polystyrene microspheres (MS) are obtained from Bangs Laboratories Inc. and dye (NeutrAvidin Dylight 650 (NA-Dy), 1 mg/mL) was purchased from Thermo Scientific. Five hundred microliters of NA-Dy solution (100 μg/mL) was prepared with PBS buffer and 50 μL of microspheres in storage buffer (100 mM Borate, pH 8.5 + 0.01% BSA +0.05% Tween® 20 + 10 mM EDTA + ≤0.1% NaN3) was mixed with the NA-Dy solution in a 1:10 ratio. The mixture was incubated on a shaker for 30 minutes (or 0.5–2 hrs, depending on the concentration) and covered by aluminum foil. Subsequently, the solution was centrifuged at 3000 rpm for 2 min and re-suspended with the buffer using a vortex mixer. The procedure was repeated 4 times. Finally, a 1 μL drop from the solution was cast onto the glass/plasmonic substrate for further experimentation.
3
Sorting Antigen-Specific Memory B Cells
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To sort antigen-specific cells, tetramers were generated as described in Franz et al. (52 (link)). A mix of Neutravidin (Neutravidin DyLight650, Thermo Scientific) and biotinylated peptide was incubated in the dark on ice for 30 min. Aggregates were removed by high speed centrifugation. B cells from acute Borreliosis patients were stained for 30 min with a pool of the three peptide tetramers and washed twice with FACS buffer (PBS, 2% FBS). The following antibodies were used to distinguish the different memory B cell subpopulations and to gate out monocytes, T-cells and dead cells: CD14-FITC, CD3-FITC (Immunotools), IgD-BV421, CD27-PECF594 (BD Horizon), CD19-BV605 (BDPharmingen), and live/dead marker. CD20-Biotin (Immunotools) was used as a compensation control for Neutravidin. Single cells were sorted on a FACSAria SORP machine (BD Biosciences) into 96-well PCR plates (Eppendorf) containing 5μl of 0.5% PBS, 10 mM DTT (Invitrogen), and 5U Recombinant RNasin® Ribonuclease Inhibitor (Promega) per well (53 (link)). The plate holder of the sorter was kept at 4°C throughout the sorting procedure. Random, negative and tetramer positive CD19+CD27+CD14−, CD3−, Hoechst− B cells were sorted into 96-well plates, which were immediately put on dry ice and stored at −80°C.
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