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Inherited Differences in Alpha1-Antitrypsin
Published in Stephen D. Litwin, Genetic Determinants of Pulmonary Disease, 2020
The commonly used method for phenotyping is starch gel electrophoresis [72]. For most phenotypes this method is sufficient. But for the recognition of heterozygotes for Piz and other low-concentration variants, starch gel electrophoresis followed by crossed Immunoelectrophoresis is necessary. However, even with the combination of these two procedures, hypomorphic variants with an electric charge similar to M cannot be recognized. Isoelectric focusing [114-118] is a very good alternate procedure that has several advantages over starch gel electrophoresis: it is less time-consuming, it avoids the uncertainties of the quality of hydrolyzed starch, 20-25 serum samples can be handled on one gel, and an immunologic identification of heterozygotes for PiZ is often unnecessary although in some instances such a confirmation is called for. As has been shown in detail, isoelectric focusing appears to be the only method that can detect the "subtype" of PiM, PiM1 [83].
Red Cells Containing Unstable Hemoglobin Variants
Published in Ronald L. Nagel, Genetically Abnormal Red Cells, 2019
Mansouri and Winterhalter26 have demonstrated that the alpha chains are a preferred site for autooxidation. Isoelectric focusing is an excellent tool to separate and quantitate the difference forms of the autoxidation process.
Application of Genomic, Proteomic, and Metabolomic Technologies to the Development of Countermeasures against Chemical Warfare Agents
Published in Brian J. Lukey, James A. Romano, Salem Harry, Chemical Warfare Agents, 2019
Jennifer W. Sekowski, James F. Dillman III
The traditional workhorse technique of proteomics is 2D-PAGE (or 2DE) (Bjellqvist et al., 1982; Klose, 1975; O’Farrell, 1975). In this method, proteins in a mixture are separated first by isoelectric focusing. In this step, the proteins migrate in a liquid or gel matrix by electric current along a pH gradient (isoelectric focusing gradient) to a point where their charge is neutral. The point along the pH gradient where a protein has a neutral charge is termed the isoelectric point (pI). In the second dimension, the proteins, held at their isoelectric points, are separated by mass. The separated polypeptides are then stained (by, e.g., silver, Coomassie blue, or Sypro Ruby) in the gel and located by their isoelectric point and mass. Identification can be made by Western blotting (in the case where an antibody is available for a protein of interest) or staining and excision (manual or robotic) followed by MS in the case where a protein is unknown. Western blotting can be a useful method of identifying large shifts in protein abundance or shifts in isoelectric point, if antibodies that recognize the proteins of interest are available. In most cases of discovery, however, shifts in either intensity or position on the gel are detected by staining all proteins and using image analysis software to identify differences in the position and abundance of spots. After the locations of these spots of interest are mapped, the spots can be excised manually or robotically for identification by MS.
A versatile design platform for glycoengineering therapeutic antibodies
Published in mAbs, 2022
Seth D. Ludwig, Zachary J. Bernstein, Christian Agatemor, Kris Dammen-Brower, Jeffrey Ruffolo, Jonah M. Rosas, Jeremy D. Post, Robert N. Cole, Kevin J. Yarema, Jamie B. Spangler
The diluted HEK 293 F or CHO-S cells and 40 mL of DNA/PEI mixture or 40 mL DNA/PBAE 4-4-6 per liter of cells were added to a shaking flask and incubated at 37°C and 5% CO2 with rotation at 125 rpm for 5 days. Secreted antibodies were purified from cell supernatants 5 days post-transfection via protein G agarose (Thermo Fisher Scientific) affinity chromatography followed by SEC using a Superdex 200 Increase 10/300 GL column (Cytiva) on a fast protein liquid chromatography (FPLC) instrument, equilibrated in HEPES-buffered saline (HBS, 150 mM NaCl in 10 mM HEPES pH 7.3). Purity was verified by SDS-PAGE analysis. Dynamic light scattering chromatograms of antibodies were collected using a Zetasizer Pro (Malvern). Native isoelectric focusing was performed using Criterion IEF (Bio-Rad) gels according to the manufacturer’s protocol. Antibody melt curves were obtained using a Protein Thermal Shift Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Melting temperatures were determined by maxima of the differential melt-curve.
Twelve Cases of Hb Manitoba [α102(G9)Ser→Arg]: the Fluctuation in the Variant Expression
Published in Hemoglobin, 2020
Mohamed S.M. Khalil, Adele T. Timbs, Shirley J. Henderson, Anna Schuh, John M. Old
This study was carried out at the National Haemoglobinopathy Reference Laboratory, Haemophilia Centre, Churchill Hospital, Oxford, UK, in 2009. Cases with abnormal hemoglobins (Hbs) have been sent from other UK hematology departments for further investigation. All specimens were analyzed on the VARIANT II™ high performance liquid chromatography (HPLC) system using the Thalassemia Short Program (Bio-Rad Laboratories, Hercules, CA, USA), and the retention time (RT) for each Hb variant was noted. All specimens were subjected to isoelectric focusing (IEF) [9]. α-Globin chain variants were selected by their percentage, RT and IEF according to our reference laboratory’s own database. Sequencing of α-globin genes was done according to a standard operating procedure. The α1 and α2 genes were amplified in separate reactions. Polymerase chain reaction (PCR) was carried out using a Qiagen multiplex kit (Qiagen GmbH, Hilden, Germany; http://www.qiagen.com). Cycle sequencing (forward and reverse) of the product for each gene was then performed using a CEQTM DTCS Quick Start kit [Beckman Coulter (UK) Ltd., High Wycombe, Buckinghamshire, UK]. The products were processed according to the Beckman Coulter CEQ DNA analysis system user manual (http://www.beckmancoulter.com) and the sequence analyzed on a Beckman Coulter CE8000 Genetic Analysis System. Detailed steps were previously published by Khalil et al. [10].
Update on the proteomics of male infertility: A systematic review
Published in Arab Journal of Urology, 2018
Manesh Kumar Panner Selvam, Ashok Agarwal
Previously, identification and quantification of multiple proteins at a single time were a challenging task. Development and introduction of new advanced tools and techniques have revolutionised the field of proteomics. In general, the protein techniques are classified into separation and identification techniques. Conventional separation techniques include separation of proteins in a given sample by sodium dodecyl sulphate (SDS)–PAGE based on their molecular weight. Similarly, two dimensional (2D)-gel electrophoresis is widely used for quantitative and qualitative proteins in a much more efficient manner based on the isoelectric focusing point and molecular weight of proteins. However, this technique is unable to generate a complete profile of all the proteins and is not sensitive enough to detect low-abundance proteins.