Dylight650 nhs ester

TDP-43-MBP-His₆ was labeled with Alexa Fluor 488 C5 maleimide (Thermo Fisher) and monoclonal TDP-43 antibodies were conjugated with DyLight™ 650 NHS Ester (Thermo Fisher), both at a low (~ 0.01–0.05) labelling efficiency to not interfere with condensate formation. Following manufacturer´s instructions, TDP-43-MBP-His₆ was mixed with the Alexa Fluor reagent in a 100:1 or 20:1 protein:fluorescent dye molar ratio and kept in the dark for 2 h at RT. Monoclonal antibodies and DyLight dye were used in a 50:1 protein:fluorescent dye molar ratio and incubated for 1 h at RT protected from light. Excess dye was removed by consecutive washes with TDP-43 purification buffer (TDP-43-MBP-His₆) or PBS (monoclonal antibodies) using Amicon ultra centrifugal filters (Merck, Germany) and protein concentrations were determined. Labeled proteins were used for confocal microscopy, aggregation, and phase separation assays, respectively. For flow-cytometry based uptake assays, TDP-43-MBP-His₆ was labeled with pHrodo™ iFL Green STP Ester (Thermo Fisher) at a labeling efficiency of ~ 0.7–1.0. The labeling procedure was carried out as described above with a protein:fluorescent dye molar ratio of 1:1 and an incubation step of 1 h.

The PBAEs were synthesized using two-step Aza-Michael addition synthesis. First, to generate the PBAE backbone, the Bisphenol A glycerolate diacrylate and 6-amino-1-hexanol (Sigma Aldrich) were dissolved in DMSO at a molar of 1.15 :1 for 23 hours at 90 °C. Next, the PBAE backbone was end-capped with PEI and modified using PDFO at 40 °C for 20 h. Before use, the PBAE polymer was lyophilized to remove the DMSO and dissolved in 25 mM HEPES buffer (pH = 7.4). To label the PBAE nanoparticle, the DyLight 650 NHS ester (ThermoFisher Scientific) was mixed with the nanoparticle at a mass ratio of 1: 100. Foxf1 plasmid was generated by cloning DNA (3HA-RFP-Foxf) into the Enhanced Episomal Vectors (EEV) empty plasmid vectors (SBI). The PBAE polymer (300 µg) were used to encapsulate 40 µg of plasmid DNA (EEV-Foxf1 or EEV-empty). The size distribution and the surface potential distribution were determined by Dynamic light scattering (DLS). Nanoparticles (250 µl) were delivered to mice via tail vein or eye vein on day 0 or day 7 after bleomycin administration.

Microarray manufacturing was performed in our lab using a noncontact microarray printer (SciFLEXARRAYER S3; Scienion, Berlin, Germany). The HTRA2 antibodies (Proteintech GmbH; Manchester, UK) were spotted onto nitrocellulose-coated microarray slides (AVID Oncyte, NC 16 Pad slides; Grace Bio-Labs, Bend, OR, USA) in triplicate. An amount of 40 µg of cell lysates from R28 cells exposed to glutamate and H₂O₂ as well as untreated controls were prepared in labeling buffer (0.05 M sodium borate buffer, pH 8.5) and subsequently labeled with a fluorescent dye (DyLight 650 NHS Ester; Thermo Fisher Scientific, Rockford, IL, USA). Samples were incubated with 1 µL of dye overnight at 4 °C in the dark. In addition, a negative control containing only the labeled buffer as well as a positive control comprising a pooled sample were also included in this experiment. To stop the labeling reaction, 10 µL of quenching solution (Tris-HCl, pH 8.8) was added to the samples and incubated for 30 min at room temperature in the dark. Unbound dye was removed using Zeba desalting plates (Zeba Spin Desalting Plates, 7k MWCO; Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s protocol.

Induction of gene expression and scFv surface presentation was achieved by inoculation of yeast cells in Synthetic Galactose minimal medium with Casein Amino Acids (SG-CAA) at an OD₆₀₀ of 1.0 and incubation overnight at 30°C and 180 rpm. For library sorting, cells were harvested by centrifugation and washed with PBS+0.1% (w/v) BSA (PBS-B). Antigen staining was conducted with DyLight650™-labelled IgM from human serum (Sigma Aldrich) conjugated beforehand using 5-fold excess of DyLight650™ NHS Ester (Thermo Fisher Scientific). Simultaneously, staining for surface presentation using anti-cMyc antibody FITC-conjugated (Miltenyi Biotec; diluted 1:50) was performed for 30 min on ice. After another PBS-B washing step, the yeast library was screened using BD Influx cell sorter with corresponding BD FACS Sortware v1.0.

4D5scFv-PE40 was labeled with fluorescein isothiocyanate (FITC, Thermo Scientific) or DyLight650 NHS Ester (Thermo Scientific), that are widely used amino-reactive dyes. These dyes contain isothiocyanate or N-hydroxysuccinimide (NHS) ester, respectively, reacting with primary amines in proteins. Prior to labeling reaction 4D5scFv-PE40 was desalted on Sephadex G-25 column (PD SpinTrap G-25, GE Healthcare) equilibrated with borate buffer (400 mM H₃BO₃, 70 mM Na₂B₄O₇, pH 8.0). For FITC labeling, the protein was incubated with 5-fold molar excess of FITC dissolved in DMSO (Thermo Scientific) for 2 h at room temperature in the dark. For DyLight650 labeling, the protein was incubated with 7-fold molar excess of DyLight650 dissolved in DMSO (Thermo Scientific) for 1 h at room temperature in the dark. To remove unbound dye the reaction mixture was then desalted on Sephadex G-25 column (PD SpinTrap G-25, GE Healthcare) equilibrated with PBS.