Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes

Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From th...
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Autor*in:	Debaize, Lydie [verfasserIn] Jakobczyk, Hélène Rio, Anne-Gaëlle Gandemer, Virginie Troadec, Marie-Bérengère

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2017

Schlagwörter:	Fusion gene Fusion protein PLA Proximity ligation assay Chromosomal rearrangement B-cells Non-adherent cells

Anmerkung:	© The Author(s). 2017

Übergeordnetes Werk:	Enthalten in: Molecular cytogenetics - London : BioMed Central, 2008, 10(2017), 1 vom: 20. Juli
Übergeordnetes Werk:	volume:10 ; year:2017 ; number:1 ; day:20 ; month:07

Links:	Volltext

DOI / URN:	10.1186/s13039-017-0328-2

Katalog-ID:	SPR029583616

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520			\|a Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.
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allfields	10.1186/s13039-017-0328-2 doi (DE-627)SPR029583616 (SPR)s13039-017-0328-2-e DE-627 ger DE-627 rakwb eng Debaize, Lydie verfasserin aut Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. Fusion gene (dpeaa)DE-He213 Fusion protein (dpeaa)DE-He213 PLA (dpeaa)DE-He213 Proximity ligation assay (dpeaa)DE-He213 Chromosomal rearrangement (dpeaa)DE-He213 B-cells (dpeaa)DE-He213 Non-adherent cells (dpeaa)DE-He213 Jakobczyk, Hélène aut Rio, Anne-Gaëlle aut Gandemer, Virginie aut Troadec, Marie-Bérengère (orcid)0000-0003-2668-9670 aut Enthalten in Molecular cytogenetics London : BioMed Central, 2008 10(2017), 1 vom: 20. Juli (DE-627)562079963 (DE-600)2420849-8 1755-8166 nnns volume:10 year:2017 number:1 day:20 month:07 https://dx.doi.org/10.1186/s13039-017-0328-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 20 07
spelling	10.1186/s13039-017-0328-2 doi (DE-627)SPR029583616 (SPR)s13039-017-0328-2-e DE-627 ger DE-627 rakwb eng Debaize, Lydie verfasserin aut Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. Fusion gene (dpeaa)DE-He213 Fusion protein (dpeaa)DE-He213 PLA (dpeaa)DE-He213 Proximity ligation assay (dpeaa)DE-He213 Chromosomal rearrangement (dpeaa)DE-He213 B-cells (dpeaa)DE-He213 Non-adherent cells (dpeaa)DE-He213 Jakobczyk, Hélène aut Rio, Anne-Gaëlle aut Gandemer, Virginie aut Troadec, Marie-Bérengère (orcid)0000-0003-2668-9670 aut Enthalten in Molecular cytogenetics London : BioMed Central, 2008 10(2017), 1 vom: 20. Juli (DE-627)562079963 (DE-600)2420849-8 1755-8166 nnns volume:10 year:2017 number:1 day:20 month:07 https://dx.doi.org/10.1186/s13039-017-0328-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 20 07
allfields_unstemmed	10.1186/s13039-017-0328-2 doi (DE-627)SPR029583616 (SPR)s13039-017-0328-2-e DE-627 ger DE-627 rakwb eng Debaize, Lydie verfasserin aut Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. Fusion gene (dpeaa)DE-He213 Fusion protein (dpeaa)DE-He213 PLA (dpeaa)DE-He213 Proximity ligation assay (dpeaa)DE-He213 Chromosomal rearrangement (dpeaa)DE-He213 B-cells (dpeaa)DE-He213 Non-adherent cells (dpeaa)DE-He213 Jakobczyk, Hélène aut Rio, Anne-Gaëlle aut Gandemer, Virginie aut Troadec, Marie-Bérengère (orcid)0000-0003-2668-9670 aut Enthalten in Molecular cytogenetics London : BioMed Central, 2008 10(2017), 1 vom: 20. Juli (DE-627)562079963 (DE-600)2420849-8 1755-8166 nnns volume:10 year:2017 number:1 day:20 month:07 https://dx.doi.org/10.1186/s13039-017-0328-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 20 07
allfieldsGer	10.1186/s13039-017-0328-2 doi (DE-627)SPR029583616 (SPR)s13039-017-0328-2-e DE-627 ger DE-627 rakwb eng Debaize, Lydie verfasserin aut Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. Fusion gene (dpeaa)DE-He213 Fusion protein (dpeaa)DE-He213 PLA (dpeaa)DE-He213 Proximity ligation assay (dpeaa)DE-He213 Chromosomal rearrangement (dpeaa)DE-He213 B-cells (dpeaa)DE-He213 Non-adherent cells (dpeaa)DE-He213 Jakobczyk, Hélène aut Rio, Anne-Gaëlle aut Gandemer, Virginie aut Troadec, Marie-Bérengère (orcid)0000-0003-2668-9670 aut Enthalten in Molecular cytogenetics London : BioMed Central, 2008 10(2017), 1 vom: 20. Juli (DE-627)562079963 (DE-600)2420849-8 1755-8166 nnns volume:10 year:2017 number:1 day:20 month:07 https://dx.doi.org/10.1186/s13039-017-0328-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 20 07
allfieldsSound	10.1186/s13039-017-0328-2 doi (DE-627)SPR029583616 (SPR)s13039-017-0328-2-e DE-627 ger DE-627 rakwb eng Debaize, Lydie verfasserin aut Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes 2017 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Author(s). 2017 Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. Fusion gene (dpeaa)DE-He213 Fusion protein (dpeaa)DE-He213 PLA (dpeaa)DE-He213 Proximity ligation assay (dpeaa)DE-He213 Chromosomal rearrangement (dpeaa)DE-He213 B-cells (dpeaa)DE-He213 Non-adherent cells (dpeaa)DE-He213 Jakobczyk, Hélène aut Rio, Anne-Gaëlle aut Gandemer, Virginie aut Troadec, Marie-Bérengère (orcid)0000-0003-2668-9670 aut Enthalten in Molecular cytogenetics London : BioMed Central, 2008 10(2017), 1 vom: 20. Juli (DE-627)562079963 (DE-600)2420849-8 1755-8166 nnns volume:10 year:2017 number:1 day:20 month:07 https://dx.doi.org/10.1186/s13039-017-0328-2 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 10 2017 1 20 07
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author	Debaize, Lydie
spellingShingle	Debaize, Lydie misc Fusion gene misc Fusion protein misc PLA misc Proximity ligation assay misc Chromosomal rearrangement misc B-cells misc Non-adherent cells Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
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topic_title	Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes Fusion gene (dpeaa)DE-He213 Fusion protein (dpeaa)DE-He213 PLA (dpeaa)DE-He213 Proximity ligation assay (dpeaa)DE-He213 Chromosomal rearrangement (dpeaa)DE-He213 B-cells (dpeaa)DE-He213 Non-adherent cells (dpeaa)DE-He213
topic	misc Fusion gene misc Fusion protein misc PLA misc Proximity ligation assay misc Chromosomal rearrangement misc B-cells misc Non-adherent cells
topic_unstemmed	misc Fusion gene misc Fusion protein misc PLA misc Proximity ligation assay misc Chromosomal rearrangement misc B-cells misc Non-adherent cells
topic_browse	misc Fusion gene misc Fusion protein misc PLA misc Proximity ligation assay misc Chromosomal rearrangement misc B-cells misc Non-adherent cells
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title	Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
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title_full	Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
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title_sort	optimization of proximity ligation assay (pla) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-b lymphocytes
title_auth	Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
abstract	Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. © The Author(s). 2017
abstractGer	Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. © The Author(s). 2017
abstract_unstemmed	Background Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field. © The Author(s). 2017
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title_short	Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes
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From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. Methods We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. Results We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. Conclusion The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients’ cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. 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Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes

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