China
Africa
Explore chapters and articles related to this topic
Isolation, Fractionation, and Analysis of Nonhistone Chromosomal Proteins
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
Leokadia Klyszejko-Stefanowicz, Lubomir S. Hnilica
The technique of isoelectric focusing has proved to be an invaluable adjunct to traditional electrophoretic procedures, differing from the latter in that various proteins in the sample move under the applied voltage to their isoelectric points in a preestablished pH gradient.333 This is important, especially where the desired protein must be separated from others of similar size. Isoelectric focusing relies on the generation of reproducible linear pH gradients formed by complex mixtures of amphoteric substances prepared by copolymerization of polyamines with suitable reactants to introduce acidic groups. Although a broad range of ampholytes covering pHs between 3 and 10 is quite useful, better resolution of protein components can be accomplished through the use of narrow-range ampholytes.334 Until a few years ago the choice of pH ranges for IFPA was limited by the commercially available pi ranges of synthetic mixtures of carrier ampholytes of the Ampholine® type (e.g., Ampholine®, Servalyte®, Pharmalyte®, etc.). Today, there is much greater freedom in the choice of carrier constituents to suit the chemical predilection of the molecule of interest, the desired pH range, carrier constituent molarity, etc.316
Natural polymeric nanocarriers in malignant glioma drug delivery and targeting
Published in Journal of Drug Targeting, 2021
Yuan Gao, Rui Wang, Lixia Zhao, Anchang Liu
Gelatine is the earliest proteinaceous formulation of NPs, which can be obtained by partial catalysed hydrolysis of collagen under alkaline or acidic conditions or by enzymatic or thermal degradation of collagen. Different employed conversion methods decide different gelatine types with different physicochemical properties, like type A possessing isoelectric point (IEP) 7-9 while type B 4–5 [120]. Gelatine is a water-soluble protein, and its polypeptide is composed of repeating triplets of alanine, glycine and proline residues. The repeating ɑ-chains formed a typical triple helical structure, responsible for the high stability of gelatine [85]. Since the presence of cationic and anionic functional groups and terminal amino and carboxyl groups, gelatine serves as a poly-ampholyte in nature. Commercially, gelatine is a heterogeneous mixture of polypeptides with varying molecular weights in different ranges, presenting an average of 20–220 kDa [86,121]. Known as an abundant raw material, gelatine is non-toxic, bioactive, biodegradable and inexpensive, regarded as a promising candidate in drug delivery and controlled drug release. Up-to-now, there are many fashioned or new-fashioned delivery strategies derived from gelatine conjugated with targeting moieties, hydrophilic polymers, fluorescence dyes or different therapeutic agents without losing their desired therapeutical properties [122].
Nidogen-1 is a novel extracellular ligand for the NKp44 activating receptor
Published in OncoImmunology, 2018
Silvia Gaggero, Maurizio Bruschi, Andrea Petretto, Monica Parodi, Genny Del Zotto, Chiara Lavarello, Carola Prato, Laura Santucci, Alessandra Barbuto, Cristina Bottino, Giovanni Candiano, Alessandro Moretta, Massimo Vitale, Lorenzo Moretta, Claudia Cantoni
Concentrated HEK293T-SN and HEK293T-SN-biot (300 μg for Western blot and 600 μg for preparative gels) were solubilized in the reduction/alkylation solution containing 8 M urea, 4% CHAPS, 5 mM tributylphosphine (TBP), 20 mM iodoacetamide (IAA), 40 mM Tris, and 0.1 mM EDTA for 1 h. To prevent over-alkylation during the isoelectro focusing (IEF) step, excess of IAA was neutralized by adding an equimolar amount of DTT. Finally, samples were dissolved in the focusing/re-hydration solution, i.e. 7 M urea, 2 M thiourea, 4% CHAPS, and 15 mM dithioerythritol (DTE) and a 0.6% (v/v) carrier ampholyte cocktail, containing 40% of the pH 3.5–10 and 60% of the pH 4–8 intervals (BDH Biochemical, 44430 2F) and loaded onto home-made non-linear pH 3–10 strips.72 After IEF runs, the strips were equilibrated in 6 M Urea, 50 mM Tris-HCl pH 8.8, 2% (w/v) SDS, 30% (v/v) glycerol, and traces of bromophenol blue; the proteins were separated using a SDS-PAGE (T% 8–16) and transferred onto nitrocellulose membranes (Protran BA85, Whatman, 10402588) with a semidry system. The membranes were saturated with 3% w/v polyvinyl-pyrrolidone (PVP) in TBS and incubated overnight separately with NKp44Fc, NKp30Fc, or NKp46Fc in 3% w/v BSA in TBS-Tween 0.15% v/v (TBS-T). Membranes were then rinsed in TBS-T and incubated with anti-human IgG HRP-conjugated mAb. HEK293T-SN-biot was subjected to the same procedure and immunoblotted with Neutravidin-HRP (ThermoFisher, 31001). For preparative experiments, SDS-gels were stained with “blue silver” colloidal Coomassie.73 Images were digitalized using ChemiDoc Touch (Bio-Rad) and analyzed with PDQuest software (Bio-Rad).
Developability assessment for monoclonal antibody drug candidates: a case study
Published in Pharmaceutical Development and Technology, 2021
Vivek Kumar Garripelli, Zhen Wu, Supriya Gupta
The charge variants were monitored by IcIEF on an iCE280 Analyzer (ProteinSimple, San Jose, CA). The samples were prepared at 0.6 mg/mL by diluting in an ampholyte mixture containing volume/volume 2.7% pharmalyte pH 5–8, 2.7% pharmalyte pH 8–10.5, 16.7% 8 M urea, 0.7% pI marker 5.85, 0.7% pI marker 9.46, and 30% 70 mM HCl. The samples were focused for 1 min at 1500 V and 8 min at 3000 V and imaged with a UV detector at 280 nm. The electropherogram was processed using Empower software to quantify the peak area percentages corresponding to the main charge isoform, the acidic charge isoforms, and the basic charge isoforms.