Profiling human serum apolipoproteins by gel isoelectric focusing in Immobilized pH Gradients /
Gel electrophoresis in Immobilized pH Gradients (IPG) using instrument IPGphor was applied for profiling human serum apolipoproteins. A novel solid-phase extraction technique using C18 phase -containing cartridges was successfully applied for lipoprotein desalting/delipidation. High-quality reprod...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=725921891&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Gel electrophoresis in Immobilized pH Gradients (IPG) using instrument IPGphor was applied for profiling human serum apolipoproteins. A novel solid-phase extraction technique using C18 phase -containing cartridges was successfully applied for lipoprotein desalting/delipidation. High-quality reproducible separation patterns of apolipoprotein bands using high-density lipoprotein (HDL), very-low density lipoprotein (VLDL), and total lipoprotein fraction (TLF), were obtained. Major apolipoproteins and their isoforms were resolved and identified. A computerized imaging method was developed for documentation and comparison of the gels. Using this technique, apolipoprotein bands on the gel were converted into peaks, forming IEF IPG profiles (densitograms). Numerous clinical samples from a cardiac ward were analyzed using the developed method. The technique allowed identification of abnormal serum profiles, as well as "abnormal" peaks in otherwise "normal" profiles. Apolipoproteins were identified based on comparison of the pI values derived from their band position on the gels, with those of apo standards analyzed under similar conditions, as well as based on literature data. The method proved to be reproducible, convenient, and robust, which made it suitable for clinical analysis. A method for quantification of apos on the gel was developed, based on extraction of staining dye by organic solvents from excised stained gel segments, with subsequent measurements of solution absorbance. A novel solution, containing formic acid, water and isopropanol (FWI), used previously for protein band destaining prior to protein elution from gels for mass spectrometry analysis, was applied to extract the dye from gel segments. Separate calibration curves were obtained for apolipoproteins A-I, A-II, C-III, and E. Reproducibility of the method was confirmed by running the Apo A-I calibration curve in duplicate. In order to provide a method for the identification of the unknown protein bands based on molecular weight of intact protein, proteins were extracted from gels for subsequent MALDI analysis. Two methods for protein extraction from IEF IPG gels were applied: electroelution and passive elution with organic solvents under sonication. Recovery of apos from gels using Apo A-I as a model protein was evaluated using free zone capillary electrophoresis and immunoturbidimetry. Mass spectra of apos from both unstained and stained gels were obtained. |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xvii, 177 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 163-173). |