The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Hermann Schagger - One of the best experts on this subject based on the ideXlab platform.
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high resolution clear native Electrophoresis for in gel functional assays and fluorescence studies of membrane protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Ilka Wittig, Michael Karas, Hermann SchaggerAbstract:Clear native Electrophoresis and blue native Electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native Electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native Electrophoresis. However, clear native Electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during Electrophoresis and therefore has been used rarely. To preserve the advantages of both Electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native Electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during Electrophoresis. This improved clear native Electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native Electrophoresis. We demonstrate the superiority of high resolution clear native Electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native Electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native Electrophoresis make this technique superior for functional proteomics analyses.
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high resolution clear native Electrophoresis for in gel functional assays and fluorescence studies of membrane protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Ilka Wittig, Michael Karas, Hermann SchaggerAbstract:Clear native Electrophoresis and blue native Electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native Electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native Electrophoresis. However, clear native Electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during Electrophoresis and therefore has been used rarely. To preserve the advantages of both Electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native Electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during Electrophoresis. This improved clear native Electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native Electrophoresis. We demonstrate the superiority of high resolution clear native Electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native Electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native Electrophoresis make this technique superior for functional proteomics analyses. Molecular & Cellular Proteomics 6:1215–1225, 2007.
Xavier Bossuyt - One of the best experts on this subject based on the ideXlab platform.
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automated serum protein Electrophoresis by capillarys
Clinical Chemistry and Laboratory Medicine, 2003Co-Authors: Xavier Bossuyt, Norbert Blanckaert, Benedicte Lissoir, Godelieve Marien, Diane Maisin, Jozef Vunckx, Pierre WallemacqAbstract:Capillary zone Electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone Electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel Electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel Electrophoresis, Capillarys, capillary Electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel Electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel Electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel Electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel Electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone Electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum Electrophoresis.
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false negative results in detection of monoclonal proteins by capillary zone Electrophoresis a prospective study
Clinical Chemistry, 2001Co-Authors: Xavier Bossuyt, Godelieve MarienAbstract:Over the last 5 years, capillary zone Electrophoresis (CZE) with the use of fused-silica capillaries has been increasingly introduced in clinical laboratories for routine serum-protein Electrophoresis (1). The multichannel, automated Paragon CZE 2000 instrument (seven capillaries in parallel; Beckman Coulter) represented an especially attractive alternative to time-consuming manual techniques. CZE has been documented to perform reliably for the analysis of serum proteins and for the detection of monoclonal components (2)(3). We reported (4) that the sensitivity of the Paragon CZE 2000 system for the detection of monoclonal components (93%) was superior to the sensitivity of cellulose acetate gel Electrophoresis (74%) and agarose gel Electrophoresis (86%). In a prospective study, Katzman et al. (5) reported sensitivities of 95% and 91% for seven-capillary Electrophoresis and agarose gel Electrophoresis, respectively. When compared with agarose gel Electrophoresis, CZE was able to detect more low-concentration IgA monoclonal components or light chains that were hidden in agarose gel Electrophoresis because of comigration with transferrin or C3 (6). Paraproteins that are missed by CZE are typically very low-concentration monoclonal components that are also missed by agarose gel Electrophoresis, but not by immunofixation. Problems with the detection of monoclonal components by CZE have been described (7)(8). Using a single-capillary Beckman P/ACE instrument with a capillary and buffer different from the Paragon CZE 2000 system, Jenkins and Guerin (7) performed a prospective study on 5500 specimens in which they compared CZE with agarose gel Electrophoresis for the detection of monoclonal components. The authors identified six paraproteins that did not separate correctly on CZE. On agarose gel Electrophoresis, all of these proteins migrated in the very slow γ region. The pI …
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serum protein Electrophoresis and immunofixation by a semiautomated Electrophoresis system
Clinical Chemistry, 1998Co-Authors: Xavier Bossuyt, Ann Bogaerts, Gilberte Schiettekatte, Norbert BlanckaertAbstract:Semiautomated agarose Electrophoresis and immunofixation performed with Hydrasys-Hyrys ™ (Sebia) were compared with conventional, manually performed methods, including cellulose acetate Electrophoresis, immunoElectrophoresis, and immunofixation. Reference intervals for agarose Electrophoresis with Hydrasys-Hyrys were determined. Within-run imprecision (CV) for fraction quantitation with the semiautomated system was between 1% (albumin) and 4.5% (β-globulin). Total imprecision (CV) was between 2.7% (albumin) and 7.3% (β-globulin). Semiautomated agarose Electrophoresis showed linear correlation with cellulose acetate Electrophoresis. Thirty-four specimens with monoclonal components were analyzed by manual immunoElectrophoresis and immunofixation and by Hydrasys. In one case, a light-chain disease was missed with Hydrasys when the sample was diluted 1:3 (the routine dilution) but not when the sample was assayed undiluted. In another case, the Hydrasys system revealed a small IgGλ monoclonal component in addition to the IgA monoclonal component detected by the manual methods. In the other cases, no differences between the manual methods and the semiautomated method were seen with respect to paraprotein identification.
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detection and classification of paraproteins by capillary immunofixation subtraction
Clinical Chemistry, 1998Co-Authors: Xavier Bossuyt, Ann Bogaerts, Gilberte Schiettekatte, Norbert BlanckaertAbstract:A selection of 58 specimens with a monoclonal component identified by immunoElectrophoresis and/or immunofixation was analyzed with the immunosubtraction procedure on the Paragon 2000 capillary Electrophoresis system. The capillary system detected 93% of the paraproteins and, using immunosubtraction, correctly identified 91% of the paraproteins. Paraproteins that were detected by immunofixation and/or immunoElectrophoresis but not by capillary Electrophoresis were also missed by agarose Electrophoresis and cellulose acetate Electrophoresis. Cellulose acetate Electrophoresis was the least sensitive method for detection of paraproteins. Only 74% of the monoclonal components were detected by this technique, whereas 86% were revealed by agarose Electrophoresis. In addition to monoclonal paraproteins, we also studied biclonal paraproteins and oligoclonal banding. Capillary Electrophoresis and immunosubtraction correctly detected and identified three specimens containing biclonal paraproteins. In one specimen, capillary zone Electrophoresis detected only one band, whereas agarose gel Electrophoresis detected two bands. The sensitivity for detection and identification of oligoclonal banding by capillary Electrophoresis was inferior to immunofixation.
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serum protein Electrophoresis by cze 2000 clinical capillary Electrophoresis system
Clinical Chemistry, 1998Co-Authors: Xavier Bossuyt, Ann Bogaerts, Gilberte Schiettekatte, Norbert BlanckaertAbstract:We compared the automated Paragon 2000 clinical capillary zone Electrophoresis (CZE) system with two manual methods, agarose Electrophoresis (AGE) and cellulose acetate Electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the α 1 -globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (α 1 -globulin). Total precision was between 0.8% (albumin) and 5.3% (β-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.
Ilka Wittig - One of the best experts on this subject based on the ideXlab platform.
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high resolution clear native Electrophoresis for in gel functional assays and fluorescence studies of membrane protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Ilka Wittig, Michael Karas, Hermann SchaggerAbstract:Clear native Electrophoresis and blue native Electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native Electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native Electrophoresis. However, clear native Electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during Electrophoresis and therefore has been used rarely. To preserve the advantages of both Electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native Electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during Electrophoresis. This improved clear native Electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native Electrophoresis. We demonstrate the superiority of high resolution clear native Electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native Electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native Electrophoresis make this technique superior for functional proteomics analyses.
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high resolution clear native Electrophoresis for in gel functional assays and fluorescence studies of membrane protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Ilka Wittig, Michael Karas, Hermann SchaggerAbstract:Clear native Electrophoresis and blue native Electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native Electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native Electrophoresis. However, clear native Electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during Electrophoresis and therefore has been used rarely. To preserve the advantages of both Electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native Electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during Electrophoresis. This improved clear native Electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native Electrophoresis. We demonstrate the superiority of high resolution clear native Electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native Electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native Electrophoresis make this technique superior for functional proteomics analyses. Molecular & Cellular Proteomics 6:1215–1225, 2007.
Norbert Blanckaert - One of the best experts on this subject based on the ideXlab platform.
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automated serum protein Electrophoresis by capillarys
Clinical Chemistry and Laboratory Medicine, 2003Co-Authors: Xavier Bossuyt, Norbert Blanckaert, Benedicte Lissoir, Godelieve Marien, Diane Maisin, Jozef Vunckx, Pierre WallemacqAbstract:Capillary zone Electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone Electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel Electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel Electrophoresis, Capillarys, capillary Electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel Electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel Electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel Electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel Electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone Electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum Electrophoresis.
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serum protein Electrophoresis and immunofixation by a semiautomated Electrophoresis system
Clinical Chemistry, 1998Co-Authors: Xavier Bossuyt, Ann Bogaerts, Gilberte Schiettekatte, Norbert BlanckaertAbstract:Semiautomated agarose Electrophoresis and immunofixation performed with Hydrasys-Hyrys ™ (Sebia) were compared with conventional, manually performed methods, including cellulose acetate Electrophoresis, immunoElectrophoresis, and immunofixation. Reference intervals for agarose Electrophoresis with Hydrasys-Hyrys were determined. Within-run imprecision (CV) for fraction quantitation with the semiautomated system was between 1% (albumin) and 4.5% (β-globulin). Total imprecision (CV) was between 2.7% (albumin) and 7.3% (β-globulin). Semiautomated agarose Electrophoresis showed linear correlation with cellulose acetate Electrophoresis. Thirty-four specimens with monoclonal components were analyzed by manual immunoElectrophoresis and immunofixation and by Hydrasys. In one case, a light-chain disease was missed with Hydrasys when the sample was diluted 1:3 (the routine dilution) but not when the sample was assayed undiluted. In another case, the Hydrasys system revealed a small IgGλ monoclonal component in addition to the IgA monoclonal component detected by the manual methods. In the other cases, no differences between the manual methods and the semiautomated method were seen with respect to paraprotein identification.
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detection and classification of paraproteins by capillary immunofixation subtraction
Clinical Chemistry, 1998Co-Authors: Xavier Bossuyt, Ann Bogaerts, Gilberte Schiettekatte, Norbert BlanckaertAbstract:A selection of 58 specimens with a monoclonal component identified by immunoElectrophoresis and/or immunofixation was analyzed with the immunosubtraction procedure on the Paragon 2000 capillary Electrophoresis system. The capillary system detected 93% of the paraproteins and, using immunosubtraction, correctly identified 91% of the paraproteins. Paraproteins that were detected by immunofixation and/or immunoElectrophoresis but not by capillary Electrophoresis were also missed by agarose Electrophoresis and cellulose acetate Electrophoresis. Cellulose acetate Electrophoresis was the least sensitive method for detection of paraproteins. Only 74% of the monoclonal components were detected by this technique, whereas 86% were revealed by agarose Electrophoresis. In addition to monoclonal paraproteins, we also studied biclonal paraproteins and oligoclonal banding. Capillary Electrophoresis and immunosubtraction correctly detected and identified three specimens containing biclonal paraproteins. In one specimen, capillary zone Electrophoresis detected only one band, whereas agarose gel Electrophoresis detected two bands. The sensitivity for detection and identification of oligoclonal banding by capillary Electrophoresis was inferior to immunofixation.
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serum protein Electrophoresis by cze 2000 clinical capillary Electrophoresis system
Clinical Chemistry, 1998Co-Authors: Xavier Bossuyt, Ann Bogaerts, Gilberte Schiettekatte, Norbert BlanckaertAbstract:We compared the automated Paragon 2000 clinical capillary zone Electrophoresis (CZE) system with two manual methods, agarose Electrophoresis (AGE) and cellulose acetate Electrophoresis (CAE). Reference intervals in healthy adults were determined for each method. When compared with AGE and CAE, CZE gave substantially higher reference values for the α 1 -globulin fraction. With CZE, within-run precision for fraction quantitation was between 0.5% (albumin) and 4.1% (α 1 -globulin). Total precision was between 0.8% (albumin) and 5.3% (β-globulin). Data obtained from CZE showed poor linear correlation with results obtained by AGE but good linear correlation with data from CAE. Analysis of serum from patients with inter alia inflammation, nephrotic syndrome, or polyclonal gammopathy showed that clinical information obtained by CZE is comparable with information obtained by AGE and CAE. We conclude that CZE offers a clinically reliable alternative to AGE and CAE and has the advantages of automation, higher precision, and faster turnaround time.
Michael Karas - One of the best experts on this subject based on the ideXlab platform.
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high resolution clear native Electrophoresis for in gel functional assays and fluorescence studies of membrane protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Ilka Wittig, Michael Karas, Hermann SchaggerAbstract:Clear native Electrophoresis and blue native Electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native Electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native Electrophoresis. However, clear native Electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during Electrophoresis and therefore has been used rarely. To preserve the advantages of both Electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native Electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during Electrophoresis. This improved clear native Electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native Electrophoresis. We demonstrate the superiority of high resolution clear native Electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native Electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native Electrophoresis make this technique superior for functional proteomics analyses.
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high resolution clear native Electrophoresis for in gel functional assays and fluorescence studies of membrane protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Ilka Wittig, Michael Karas, Hermann SchaggerAbstract:Clear native Electrophoresis and blue native Electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native Electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native Electrophoresis. However, clear native Electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during Electrophoresis and therefore has been used rarely. To preserve the advantages of both Electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native Electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during Electrophoresis. This improved clear native Electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native Electrophoresis. We demonstrate the superiority of high resolution clear native Electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I–V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native Electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native Electrophoresis make this technique superior for functional proteomics analyses. Molecular & Cellular Proteomics 6:1215–1225, 2007.
