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Biophysical and Biochemical Characterization of Peptide, Protein, and Bioconjugate Products
Published in Sandeep Nema, John D. Ludwig, Parenteral Medications, 2019
Tapan K. Das, James A. Carroll
IEF is a gel-based method which separates analytes in an immobilized pH gradient [113]. Proteins will migrate in an electric field to their isoelectric point (pI), which is the pH at which the overall charge is net neutral. Charge variants can be well separated using this technique, with resolution as high as 0.01 pH units. The resolution can be determined by the gradient used in the separation. For high-resolution separations, a very narrow pH gradient may be used with long focusing times. IEF offers a reproducible method for establishing consistency of batches with regard to charge variants, as well as a powerful method for monitoring stability of protein therapeutics. This method is tried and true, but is not highly quantitative due to the need for general protein staining and densitometry, both of which may be highly variable.
Analytical Chemistry
Published in W. M. Haynes, David R. Lide, Thomas J. Bruno, CRC Handbook of Chemistry and Physics, 2016
W. M. Haynes, David R. Lide, Thomas J. Bruno
FT-MS: Fourier transform mass spectrometry FWHM: full-width half-maximum GC: gas chromatography GC-IR: gas chromatography-infrared spectrometry GCMS: gas chromatography mass spectrometry GDL: glow discharge lamp GDMS: glow discharge mass spectrometry GE: gel electrophoresis, gradient elution GEMBE: gradient elution moving boundary electrophoresis GFAAS: graphite furnace atomic absorption spectroscopy GLC: gas-liquid chromatography GPC: gel permeation chromatography GS: Gram-Schmidt (algorithm) GSC: gas-solid chromatography GSED: gaseous secondary electron detector HCL: hollow cathode lamp HCOT: helically coiled open tubular (column) HDC: hydrodynamic chromatography HETCOR: heteronuclear correlation HETP: height equivalent of (a) theoretical plate(s) HG: hydride generation HIC: hydrophoric interaction chromatography HMBC: heteronuclear multiple-bond correlation HPAC: high-performance affinity chromatography HPIAC: high-performance immunoaffinity chromatography HPLC: high-performance liquid chromatography, high-pressure liquid chromatography HPTLC: high-performance thin-layer chromatography HRCGC: high-resolution capillary gas chromatography HRGC: high-resolution gas(-liquid) chromatography HS: head space HSA: hemispherical analyzer HSC: heteronuclear shift correlation HSQC: heteronuclear single quantum coherence IA: isocratic analysis IAC: immunoaffinity chromatography IAES: ion-excited Auger electron spectroscopy IC: ion chromatography ICP: inductively coupled plasma ICP-OES: ICP optical emission spectrometry ICR: ion cyclotron resonance IDMS: isotope dilution mass spectrometry IEC: ion-exchange chromatography IEF: isoelectric focusing IF: intermediate frequency IGC: inverse gas chromatography ILDA: intensified linear diode array IMAC: immobilized metal-ion affinity chromatography INADEQUATE: incredible natural abundance double-quantum transfer experiment INEPT: insensitive nuclei enhancement by polarization transfer INAA: instrumental neutron activation analysis IP: ion pairing IPC: ion-pair chromatography IPG: immobilized pH gradient IPMA: ion probe microanalysis IR: infrared (spectrophotometry) IRN: indicator radionuclide(s) IRS: internal reflection spectroscopy ISCA: ionization spectroscopy for chemical analysis ISE: ion selective electrode ISP: ion spray ISS: ion scattering spectrometry
Toxicoproteomic assessment of liver responses to acute pyrrolizidine alkaloid intoxication in rats
Published in Journal of Environmental Science and Health, Part C, 2018
Yan-Hong Li, William Chi-Shing Tai, Imran Khan, Cheng Lu, Yao Lu, Wing-Yan Wong, Wood-Yee Chan, Wen-Luan Wendy Hsiao, Ge Lin
RTS, the reduced form of glutathione (GSH), 5,5-dithiobis-2-nitrobenzonic acid, hydrogen peroxide (H2O2), bilirubin, sulfanilic acid, dimethyl sulfoxide, and sodium nitrite were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Acrylamide mix and siLenfect were purchased from Bio-Rad (Hercules, Calif., USA). 1, 4-Dithiothreitol (DTT), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium dodecyl sulfate (SDS), and immobilized pH gradient buffer were purchased from Amersham Biosciences (Piscataway, NJ, USA). Iodoacetamide and rat endothelial cell antibody (RECA-1) (#ab9774) were purchased from Abcam (Cambridge, MA, USA). Anti-mouse IgG antibody (#7076) was purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were of high purity and commercially available.
Allergenicity assessment of fungal species using immunoclinical and proteomic techniques: a study on Fusarium lateritium
Published in International Journal of Environmental Health Research, 2020
Debarati Dey, Swati Gupta Bhattacharya
For 2D gel electrophoresis, the protein was processed through Focus Perfect 2D Clean up Kit (G Biosciences, USA) and reconstituted freshly in 125 μl IEF buffer. One percent immobilized pH gradient (IPG) pH 3–10 linear buffer (v/v) (GE Healthcare, Uppsala, Sweden), 25 mM DTT, and traces of Bromophenol blue were added. Rehydration loading was done with 7 cm Immobiline Dry Strips (Amersham Biosciences, San Francisco, CA, USA) in a re-swelling tray for overnight. Isoelectric focusing was done using EttanIPGphor 3 isoelectric focusing system (GE Healthcare) in gradient mode. Next day, strips were run in 12% SDS-PAGE following equilibration. Gels were run in triplicates and stained with CBB-R250.