The Experts below are selected from a list of 17433 Experts worldwide ranked by ideXlab platform
Kristen M Johansen - One of the best experts on this subject based on the ideXlab platform.
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preparation of drosophila polytene chromosome squashes for Antibody Labeling
Journal of Visualized Experiments, 2010Co-Authors: Weili Cai, Jack Girton, Jorgen Johansen, Ye Jin, Kristen M JohansenAbstract:Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae. In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies. The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins. Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the Labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for Antibody Labeling.
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polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in drosophila using antibodies
Methods, 2009Co-Authors: Kristen M Johansen, Weili Cai, Jack Girton, Huai Deng, Weiguo Zhang, Xiaomin Bao, Jorgen JohansenAbstract:The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the Labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable Antibody Labeling and preservation of high resolution chromatin structure.
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skeletor a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix
Journal of Cell Biology, 2000Co-Authors: Diana L Walker, Jorgen Johansen, Ye Jin, Dong Wang, Uttama Rath, Yanming Wang, Kristen M JohansenAbstract:A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor Antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor Antibody Labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.
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Antibody identification, chromosome map assignment, and sequence analysis of a Rab escort protein homolog in Drosophila1.
Biochimica et Biophysica Acta, 1999Co-Authors: Hao Dong, Jorgen Johansen, Kristen M JohansenAbstract:Using a polyclonal antiserum a cDNA encoding a Rab escort protein (REP) homolog in Drosophila has been identified and sequenced. The gene encodes a 511 residue protein with a predicted molecular mass of 56 855 Da. Antibody Labeling demonstrates that Drosophila REP protein is present in the early embryo and that it is being apportioned uniformly throughout the embryo in a process likely to be linked to the syncytial nuclear divisions. In situ hybridization to polytene chromosomes reveals that the Drosophila REP gene is located in the 56E region on the second chromosome. Drosophila REP is the first invertebrate REP homolog to be identified and characterized.
Hanna Bjorkelund - One of the best experts on this subject based on the ideXlab platform.
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evaluating real time immunohistochemistry on multiple tissue samples multiple targets and multiple Antibody Labeling methods
BMC Research Notes, 2013Co-Authors: Louise Dubois, Karl Andersson, Anna Asplund, Hanna BjorkelundAbstract:Background Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of Antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.
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evaluating real time immunohistochemistry on multiple tissue samples multiple targets and multiple Antibody Labeling methods
BMC Research Notes, 2013Co-Authors: Louise Dubois, Karl Andersson, Anna Asplund, Hanna BjorkelundAbstract:Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of Antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method. Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the Antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes. It was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.
Jorgen Johansen - One of the best experts on this subject based on the ideXlab platform.
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preparation of drosophila polytene chromosome squashes for Antibody Labeling
Journal of Visualized Experiments, 2010Co-Authors: Weili Cai, Jack Girton, Jorgen Johansen, Ye Jin, Kristen M JohansenAbstract:Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae. In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies. The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins. Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the Labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for Antibody Labeling.
-
polytene chromosome squash methods for studying transcription and epigenetic chromatin modification in drosophila using antibodies
Methods, 2009Co-Authors: Kristen M Johansen, Weili Cai, Jack Girton, Huai Deng, Weiguo Zhang, Xiaomin Bao, Jorgen JohansenAbstract:The giant polytene chromosomes from Drosophila third instar larval salivary glands provide an important model system for studying the architectural changes in chromatin morphology associated with the process of transcription initiation and elongation. Especially, analysis of the heat shock response has proved useful in correlating chromatin structure remodeling with transcriptional activity. An important tool for such studies is the Labeling of polytene chromosome squash preparations with antibodies to the enzymes, transcription factors, or histone modifications of interest. However, in any immunohistochemical experiment there will be advantages and disadvantages to different methods of fixation and sample preparation, the relative merits of which must be balanced. Here we provide detailed protocols for polytene chromosome squash preparation and discuss their relative pros and cons in terms of suitability for reliable Antibody Labeling and preservation of high resolution chromatin structure.
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skeletor a novel chromosomal protein that redistributes during mitosis provides evidence for the formation of a spindle matrix
Journal of Cell Biology, 2000Co-Authors: Diana L Walker, Jorgen Johansen, Ye Jin, Dong Wang, Uttama Rath, Yanming Wang, Kristen M JohansenAbstract:A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor Antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor Antibody Labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.
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Antibody identification, chromosome map assignment, and sequence analysis of a Rab escort protein homolog in Drosophila1.
Biochimica et Biophysica Acta, 1999Co-Authors: Hao Dong, Jorgen Johansen, Kristen M JohansenAbstract:Using a polyclonal antiserum a cDNA encoding a Rab escort protein (REP) homolog in Drosophila has been identified and sequenced. The gene encodes a 511 residue protein with a predicted molecular mass of 56 855 Da. Antibody Labeling demonstrates that Drosophila REP protein is present in the early embryo and that it is being apportioned uniformly throughout the embryo in a process likely to be linked to the syncytial nuclear divisions. In situ hybridization to polytene chromosomes reveals that the Drosophila REP gene is located in the 56E region on the second chromosome. Drosophila REP is the first invertebrate REP homolog to be identified and characterized.
Zhaowei Zhang - One of the best experts on this subject based on the ideXlab platform.
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on site ultrasensitive detection paper for multiclass chemical contaminants via universal bridge Antibody Labeling mycotoxin and illegal additives in milk as an example
Analytical Chemistry, 2019Co-Authors: Guanghua Li, Wenqin Wu, Saranya Poapolathep, Amnart Poapolathep, Lin Xu, Du Wang, Xiaomei Chen, Jun Jiang, Wen Zhang, Zhaowei ZhangAbstract:Multiclass chemical contamination of food has aroused ever-increasing attention due to the increasingly common findings of the co-occurrence of multiclass contamination, such as mycotoxin (aflatoxin M1, AFM1) and illegal additive (melamine, MEL). In the present study, a rapid, ultrasensitive detection paper was developed on the basis of a unique bridge-Antibody label to realize on-site simultaneous detection of AFM1 and MEL in milk. This detection paper used the bridge-Antibody label on fluorescent particles (i.e., the fluorescent Eu nanoparticles were first conjugated with polyclonal antibodies and then with monoclonal antibodies). Dramatically enhanced sensitivity was found, probably due to the increase in immobilization of efficient monoclonal antibodies onto microspheres. Under optimal conditions, the lower limits of detection were 0.009 and 0.024 ng/mL for AFM1 and MEL in milk, respectively, in comparison with similar works. Moreover, the cutoff values were 0.4 and 150 ng/mL for AFM1 and MEL, respect...
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on site ultrasensitive detection paper for multiclass chemical contaminants via universal bridge Antibody Labeling mycotoxin and illegal additives in milk as an example
Analytical Chemistry, 2019Co-Authors: Du Wang, Saranya Poapolathep, Amnart Poapolathep, Xiaomei Chen, Zhaowei Zhang, Jun Jiang, Wen Zhang, Qi ZhangAbstract:Multiclass chemical contamination of food has aroused ever-increasing attention due to the increasingly common findings of the co-occurrence of multiclass contamination, such as mycotoxin (aflatoxin M1, AFM1) and illegal additive (melamine, MEL). In the present study, a rapid, ultrasensitive detection paper was developed on the basis of a unique bridge-Antibody label to realize on-site simultaneous detection of AFM1 and MEL in milk. This detection paper used the bridge-Antibody label on fluorescent particles (i.e., the fluorescent Eu nanoparticles were first conjugated with polyclonal antibodies and then with monoclonal antibodies). Dramatically enhanced sensitivity was found, probably due to the increase in immobilization of efficient monoclonal antibodies onto microspheres. Under optimal conditions, the lower limits of detection were 0.009 and 0.024 ng/mL for AFM1 and MEL in milk, respectively, in comparison with similar works. Moreover, the cutoff values were 0.4 and 150 ng/mL for AFM1 and MEL, respectively. The recoveries ranged from 88.7% to 105.0% for AFM1 and from 84.6% to 117.7% for MEL, with relative standard deviations (RSDs) of 0.5-9.9% during the intraday and interday experiments. Comparison experiments conducted using the detection paper, HPLC, and UPLC-MS/MS found excellent agreement in the simultaneous detection of AFM1 and MEL in milk. This proposed method can be extensively employed for simultaneous monitoring of multiclass chemical contaminants to ensure food safety.
Louise Dubois - One of the best experts on this subject based on the ideXlab platform.
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evaluating real time immunohistochemistry on multiple tissue samples multiple targets and multiple Antibody Labeling methods
BMC Research Notes, 2013Co-Authors: Louise Dubois, Karl Andersson, Anna Asplund, Hanna BjorkelundAbstract:Background Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of Antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.
-
evaluating real time immunohistochemistry on multiple tissue samples multiple targets and multiple Antibody Labeling methods
BMC Research Notes, 2013Co-Authors: Louise Dubois, Karl Andersson, Anna Asplund, Hanna BjorkelundAbstract:Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of Antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method. Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the Antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes. It was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.