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Functional Study of Lysosomal Nutrient Transporters
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Xavier Leray, Corinne Sagné, Bruno Gasnier
All steps are carried out in modified Barth’s solution (MBS: see Section 6.3.4.2) buffered with 5 mM Hepes, MES, or Bis-Tris propane adjusted to the required pH with NaOH. Uptake is measured in 24-well plates at 20°C because Xenopus oocytes are damaged at temperatures above 22°C. Each tested condition is applied to a batch of 10 oocytes to average the high intercellular variability in expression level. If oocytes have been pre-selected for expression by fluorescence microscopy (see above), this number may be reduced to five (triplicate is not enough). Oocytes are first washed in 500 µL MBS buffered at neutral pH. They are then transferred into 200 µL MBS adjusted to the desired pH (5.0–8.5) supplemented with the radiotracer (0.5 µCi/well; total concentration 10 µM or 100 µM). After the chosen duration, uptake is stopped by washing each well 2–4 times with 2 mL ice-cold MBS. Oocytes are then individually collected and lysed in NaOH 0.1 N or SDS 10% before scintillation counting.
Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life
Published in mAbs, 2019
Brian C. Mackness, Julie A. Jaworski, Ekaterina Boudanova, Anna Park, Delphine Valente, Christine Mauriac, Olivier Pasquier, Thorsten Schmidt, Mostafa Kabiri, Abdullah Kandira, Katarina Radošević, Huawei Qiu
The FcRn affinity column was adapted from Schlothauer et al. with biotinylated hFcRn on a 1 mL Streptavidin HP HiTrap column (GE Healthcare). The column was injected with 300 μg of each antibody in low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES; Sigma) pH 5.5; 150 mM NaCl) on an AKTA Pure System (AKTA). The antibodies were eluted by a pH gradient created with low and high pH buffer (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bis tris propane; Sigma) pH 9.5; 150 mM NaCl) over 30 column volumes (CV) at 0.5 mL min−1 and monitoring the absorbance and pH. The creation of a linear pH gradient (linear regression, R2 > 0.99) was achieved through the following stepwise format: 0–30% high pH buffer over 9 CV, 30–70% over 16.5 CV and 70–100% over 4.5 CV. The column was re-equilibrated with low pH buffer for subsequent runs. All variants were performed in triplicate. The FcRn affinity column elution profiles were fit to a single Gaussian distribution in Sigmaplot 11 (Systat Software, Inc.) to determine the elution volume, full width at half maximum (FWHM) and pH from at the UV280 maximum.