Rabbit skeletal muscle actin labeled with DyLight550-NHS ester (Thermo Fisher Scientific) was prepared as described previously (Yamashiro et al., 2014 (link)).
Dylight550 nhs ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
DyLight550-NHS ester is a fluorescent labeling reagent. It is used for the covalent attachment of a fluorescent dye to proteins and other biomolecules containing primary amine groups.
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Lab products found in correlation
5 protocols using dylight550 nhs ester
1
Labeling Rabbit Skeletal Actin
2
Detecting LSR/angulin-1 in pMBMEC
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The primary and secondary antibodies used in this study are described in Table S1 . IL-1β (PeproTech, Rocky Hill, NJ, USA) stimulation of pMBMEC monolayers was done for 16–20 h at a concentration of 0.05 ng/ml (IL-1 βlo) or 20 ng/ml (IL-1 βhi). Angubindin-1, used to detect LSR/angulin-1, was coupled with DyLight 550 NHS ester with the DyLight Antibody labeling kit (Thermo Fisher Scientific, Carlsbad, CA, USA), according to the manufacturer's instructions.
3
Characterization of Complement C3 Interactions
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Electrophoretic analyses were carried out according to standard protocols. Pulse-labelling with tritiated lysine was performed for 24 h by adding 0.5% (v/v) of L-[4,5-3H(N)]-Lysine (Specific Activity: 80–110 Ci/mmol) to DMEM/decomplemented FCS. Complement C3 was immunodetected using anti-C3 antibodies (Origen). Samples were prepared for mass spectrometry analyses as previously17 (link) with minor modifications. The culture medium was supplemented with 0.8 mM heavy Lysine and 0.4 mM AHA. Click chemistry was carried out using dibenzocyclooctyne (DBCO) magnetic beads according to the manufacturer’s instructions (Jena Bioscience). The beads were then extensively washed with urea and SDS and the coupling of proteins to beads was examined by addition of fluorescein-5-maleimide and SDS-PAGE. Complement C3 was fluorescently labelled by mixing 0.1 nmol of purified human C3 (Teco Medical) with 2 nmol of DyLight 550 NHS Ester (Thermo Fisher) according to the manufacturer’s instructions and excess dye was removed with spin columns (cut-off 5 k). The efficiency of the labelling was then evaluated by SDS-PAGE (Supplementary Fig. 8 ).
4
Characterization of FGFR1, HER2, and Tubulin
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The primary antibodies directed against FGFR1 (#9740) were from Cell Signaling (Danvers, MA, USA), anti-HER2 primary antibodies (sc-33684, sc-8036) were from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-tubulin primary antibodies (#T6557) were from Sigma-Aldrich (St. Louis, MO, USA). Secondary antibodies coupled to HRP were from Jackson Immuno-Research Laboratories (Cambridge, UK). Tunicamycin was from Santa Cruz Biotechnology. DyLight™ 550 NHS Ester used for fluorescent protein labeling was from Thermo Fisher Scientific (Waltham, MS, USA).
5
Purification and Characterization of fd Bacteriophages
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Wild-type fd bacteriophages were grown with ER2738 strain of E. coli as host bacteria, and were purified according to standard protocols (51) . The fd-wt (wild type) viruses are rod-like particles, monodisperse in size and shape, with contour length ℓ = 880 nm and diameter d = 7 nm. In suspension, the virus rods have been shown to behave as near-hard rods exhibiting a well-defined liquid-crystalline phase behavior (41) (39) (40) (46) . Virus suspensions were extensively dialyzed against TRIS-NaCl-HCl buffer (pH 8.2) at 110 mM of ionic strength, to ensure the electrostatic repulsion between viral particles is strongly screened (Debye screening length, λD = 0.9 nm). The rods were subsequently concentrated using ultracentrifugation and redispersed at 30-35 mg/mL in the same buffer solution. The virus concentration was determined using spectrophotometry at the peak absorption wavelength of 269 nm with an optical density of 3.84 cm 2 /mg (52) .
Fluorescently-labeled virus batches were separately prepared by conjugating Alexa Fluor 488-TFP ester (Invitrogen) or Dylight550-NHS ester (ThermoFischer) to their coat proteins. These labeled viruses were added in tracer amounts (0.001% (w/w) to 0.1% (w/w)) to the non-labeled virus batches for tracking by fluorescence microscopy.
Fluorescently-labeled virus batches were separately prepared by conjugating Alexa Fluor 488-TFP ester (Invitrogen) or Dylight550-NHS ester (ThermoFischer) to their coat proteins. These labeled viruses were added in tracer amounts (0.001% (w/w) to 0.1% (w/w)) to the non-labeled virus batches for tracking by fluorescence microscopy.
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