China
Africa
Explore chapters and articles related to this topic
Temperature Effects in Reversed-Phase Liquid Chromatography
Published in Nelu Grinberg, Peter W. Carr, Advances in Chromatography Volume 57, 2020
Anthony R. Horner, Erin P. Shields, Michael T. Rerick, Stephen G. Weber
There have been several publications demonstrating the intriguing properties of temperature-dependent stationary phases where the stationary phase itself changes at different temperatures. Almost all of these phases are based on poly(isopropyl acrylamide)242–250 or related polymers.250–257 The intriguing nature of this system is that retention (reversed phase) increases when temperature increases. For example, Cao et al.258 used a traveling cold spot to elute proteins in a preparative separation with an N-isopropyl acrylamide-based cation exchange gel (fast protein liquid chromatography, FPLC). However, the very poor plate count typically observed makes this phase less useful than it might otherwise be.
Protein Expression Methods
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Protein chromatography can be carried out using simple gravity-flow methods or complex instrumentation and at low, medium, or high pressure. The simplest systems employ a simple chromatography column connected to a fraction collector. A slightly more complicated low-pressure system uses a peristaltic pump to provide a constant flow rate and an inline UV detector to detect protein elution from the column (Figure 5.7a). Protein elution is typically detected by absorption at 220 nm of the peptide backbone, absorption at 280 nm of aromatic amino acids, or both. Medium-pressure systems, such as GE Healthcare’s fast protein liquid chromatography (FPLC) and Bio-Rad’s medium-pressure liquid chromatography (MPLC), allow for higher flow rates and computer control, and incorporate an injection valve or autoinjector.
Protocols for Key Steps in the Development of an Immunoassay
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Caroline Murphy, Richard O’Kennedy
This protocol outlines the use of Superdex® resin from GE Healthcare for the purification of antibodies by fast protein liquid chromatography (FPLC). A number of different purification resins are available from GE Healthcare; and each one is tailored for a particular application: Sephacryl or Sepharose—wide ranges of fractionation proceduresSuperdex for high-resolution purificationSephadex LH for separation of small molecules in organic solvents
Compartmentalization of therapeutic proteins into semi-crystalline PEG-PCL polymersomes
Published in Soft Materials, 2021
Juliana de Almeida Pachioni-Vasconcelos, Alexsandra Conceição Apolinário, André Moreni Lopes, Adalberto Pessoa, Leandro Ramos Souza Barbosa, Carlota de Oliveira Rangel-Yagui
Size Exclusion Chromatography was also employed to determine the proteins encapsulation for the PEG-PCL PL after purification, adapting the method reported by Bartenstein and collaborators.[17] Samples were injected into a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences, Uppsala, Sweden) and the elution was performed isocratically in a Fast Protein Liquid Chromatography (FPLC) ÄKTA Purifier (GE Healthcare Life Sciences) in 50 mM Tris-HCl buffer, pH 8.6, with 0.5 mL/minflow rate and monitoring the absorbance at 280 nm. The protein concentration was calculated by the integration of the peak corresponding to the free protein not taking into account the aggregated fraction and the initial protein solution (added during the hydration of the polymeric film).
Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli
Published in Preparative Biochemistry & Biotechnology, 2020
Inamur Rahman, Lina Fang, Zhang Wei, Xiaodong Zheng, Lian Jiazhang, Lei Huang, Zhinan Xu
The dissolved precipitated fusion protein was mixed with EK enzyme, and EK buffer (20 mM Tris HCl, 50 mM NaCl, and 2 mM CaCl2) at the ratio of 10:1:1, respectively i.e. (10 ml of the purified Scl2-M-hbFGF was mixed with 1 ml EK buffer and 1 ml of EK enzyme) to remove the Scl2-M fusion tag. The crude mixture was placed and incubated at 25 °C for 8 h. The mature recombinant hbFGF protein was further purified by ion-exchange chromatography with a cation exchange matrix CM Sepharose FF using fast protein liquid chromatography system (AKTA-FPLC, Amersham Pharmacia Biotech GE, Uppsala, Sweden). The column was pre-equilibrated with binding buffer B (20 mM Tris-HCl pH 8.1–8.2) at a flow rate of 4 mL/min. To prevent the column from unspecific protein binding, it was rinsed by buffer B until OD280 reached to the background level and the pH of the effluent was the same as binding buffer B. The bound recombinant hbFGF was eluted from the cation exchange CM Sepharose FF column by a stepwise linear-gradient wash with elution buffer C (20 mM Tris-HCl, 1 M NaCl, pH 8.1–8.2). The fractions were collected separately during elution and examined on SDS-PAGE. Finally, the fractions containing the purified hbFGF were buffer exchanged (PBS) and lyophilized for biological activity studies. All the extraction and purification processes were performed at 4 °C.
Protein a resin lifetime study: Evaluation of protein a resin performance with a model-based approach in continuous capture
Published in Preparative Biochemistry and Biotechnology, 2018
Ketki Behere, Bumjoon Cha, Seongkyu Yoon
Once the model, feed material and concentration, and stationary phases were set, the chromatography runs were performed using the Contichrom Cube lab 10 FPLC system (ChromaCon, Zurich, Switzerland). Single-column breakthrough study was performed using the twin-column CaptureSMB system, equipped with UV detectors of 280 and 300 nm. The chromatographic run utilized 6 column volumes (CVs) of equilibration buffer, 150 CVs of load, 12 CVs of wash buffer, 8 CVs of elution buffer, 10 CVs of 0.5 M NaOH sanitization buffer, assisted by ChromIQ™ operating software (ChromaCon, Zurich, Switzerland). The columns were incubated with 0.5 M NaOH for 15 hr (60 cycles of 15-min contact time) after which the column was equilibrated and the above protocol was repeated. The new resin was allowed to equilibrate for an additional 20 CVs of equilibration buffer to saturate and prepare them for the chromatography runs. PBS buffer (pH 7.32) was used for the equilibration and wash steps, and it was also used to dilute the feed material from the initial concentration of 19.1 to 1.2 g/L. Then, 0.1 M glycine at pH 2.93 was selected as the elution buffer.