Application of polyacrylamide slab gel electrophoresis to the analysis and small-scale purification of amyloid proteins

Kaplan, Batia

doi:10.1016/s0003-2670(98)00336-5

Cited by 6 publications

(3 citation statements)

References 129 publications

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“…However, the pore size of separation gels is similar, leading to a poor resolution for the complex cytosol mixture [33]. Furthermore, the low-molecular-weight proteins would easily diffuse during the electrophoresis when using the native PAGE.…”

Section: Comparison Between Native Pg-page and Page Methodsmentioning

confidence: 99%

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

et al. 2005

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The cytosolic enzymes extracted from rat hepatocytes were separated by native porous gradient-PAGE (PG-PAGE) and were detected with a sensitive and fast chemiluminescence (CL) imaging method. Several peroxidases including glutathione peroxidase, Cu/Zn-superoxidase dismutase, and some other metallo-enzymes such as catalase, carbonic anhydrase-III (CA-III) present in the cytosol of rat hepatocytes have been selectively and sensitively detected by the direct CL imaging method using the luminol-H(2)O(2) chemiluminescent reagents. All detections after PG-PAGE were completed within 9 min. The linear range for the typical metallo-enzyme, e.g., CA-III is 0.75-4.9 microg/mL, with a detection limit of 0.25 microg/mL. In comparison with the traditional CBB-R250 staining method, the detection period decreased about 70 times and the detection sensitivity improved over ten times. Furthermore, two enzymes present in rat liver cytosol were identified employing MALDI-MS analysis of the tryptic digest after PG-PAGE.

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Section: Comparison Between Native Pg-page and Page Methodsmentioning

confidence: 99%

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

et al. 2005

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“…We faced such a situation in the analysis of fresh frozen (non-fixed) tissue extracts where the content of amyloid proteins was small and the contamination by other co-extracted tissue proteins was great. 45 In fact, micropreparative slab gel SDS electrophoresis allows the efficient separation of proteins on the basis of their differences in molecular mass, but in using this method separated amyloid proteins might still be contaminated by other proteins of the same electrophoretic mobility. In cases where the amount of the available biopsy material was sufficient (such as subcutaneous fat biopsies 13 14 ), purification was achieved by combining gel filtration on a 800 × 9 mm internal diameter Sephacryl S-2000 HR (Pharmacia, Uppsala, Sweden) column with reversed phase HPLC on a 250 × 4.6 mm VYDAC 214 TPS (Hesperia, California, USA) column.…”

Section: Combined Purification Techniquesmentioning

confidence: 99%

“…Although the use of size exclusion HPLC columns could be more appropriate for small samples, our experience showed that in many instances the resolution of amyloid proteins obtained by size exclusion HPLC was less efficient than when micropreparative slab gel SDS electrophoresis was used. 45 We developed a technique that combines slab gel SDS electrophoresis (SDS-PAGE) with subsequent reversed phase HPLC, 44 48 49 and recently adapted it for the purification of AA, AL, ATTR, and Aβ proteins microextracted from non-fixed tissues. 33 34 50 Using this technique, the electrophoresed proteins were transferred to PVDF membranes, then eluted from the membranes, and finally applied to reversed phase HPLC.…”

Section: Combined Purification Techniquesmentioning

confidence: 99%

Micropurification techniques in the analysis of amyloid proteins

Kaplan

2003

Journal of Clinical Pathology

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Gel Electrophoresis in Protein and Peptide Analysis

Kaplan

2000

Encyclopedia of Analytical Chemistry

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Gel electrophoresis is a widely used method in biochemical research; a number of different forms of gel electrophoresis have been developed and applied to the analysis of proteins and peptides. The most popular form is polyacrylamide gel electrophoresis (PAGE), in which proteins are separated within a gel matrix on the basis of differences in their charge density and/or relative molecular mass (M r ). Polyacrylamide gel is also widely used as a media for the generation of pH gradients in an isoelectrofocusing (IEF) technique, where separation of proteins is due to their differences in isoelectric point (pI). Two‐dimensional (2D) electrophoresis is an extremely powerful separation technique, allowing differentiation of proteins on the basis of their pI in IEF, and their M r in sodium dodecyl sulfate (SDS)/PAGE. Agarose gels are rarely used today in the electrophoretic analysis of proteins; their utility is mainly restricted to the immunoelectrophoretic techniques, which allow the characterization of proteins by their migration in gel and immunological properties. This article provides a brief history of gel electrophoresis and describes the principles of major electrophoretic techniques. The utility of gel electrophoresis in different fields of biochemical research is also demonstrated. Topics included are analytical and small‐scale preparative separations of complex proteins samples; determination of molecular mass of the electrophoretically separated proteins and polypeptides; electrophoretic micropreparation of a protein sample for its further immunochemical, enzymatic, and chemical analysis; study of protein interactions with different ligands; examination of protein unfolding and its quaternary structure. The advantages and limitations of the described techniques are discussed.

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Application of polyacrylamide slab gel electrophoresis to the analysis and small-scale purification of amyloid proteins

Cited by 6 publications

References 129 publications

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

Direct chemiluminescent imaging detection of Cu/Zn-superoxidase dismutase, glutathione peroxidase, carbonic anhydrase-III, and catalase in rat liver cytosol separated by native porous gradient polyacrylamide gel electrophoresis

Micropurification techniques in the analysis of amyloid proteins

Gel Electrophoresis in Protein and Peptide Analysis

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