Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved reg...Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain （Vh） and light chain （Vl） were amplified, and the Vh and modified Vl were connected to single chain Fv （ScFv） by SOE-PCR （splice overlap extension PCR）. The modified ScFv was fused with green fluorescent protein （GFP） and introduced into E. coli JM109. The fusion protein induced by lPTG （Isopropylthiogalactoside） was about 57000 on a 10% SDS-PAGE gel （10% Sds Polyacrylamide Gel Electrophoresis）, and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-l lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.展开更多
The introduction of super-resolution microscopy(SRM)has significantly advanced our understanding of cellular and molecular dynamics,offering a detailed view previously beyond our reach.Implementing SRM in biophysical ...The introduction of super-resolution microscopy(SRM)has significantly advanced our understanding of cellular and molecular dynamics,offering a detailed view previously beyond our reach.Implementing SRM in biophysical research,however,presents numerous challenges.This review addresses the crucial aspects of utilizing SRM effectively,from selecting appropriate fluorophores and preparing samples to analyzing complex data sets.We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM.Emphasizing the integration of SRM with other analytical methods,we aim to overcome inherent limitations and expand the scope of biological insights achievable.By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives,we aim to empower researchers to explore complex biological processes with enhanced precision and clarity,thereby advancing the frontiers of biophysical research.展开更多
基金Project (No. 396007) supported by the National Natural ScienceFoundation of China
文摘Total RNA was isolated from the hybridoma cell line （LC- 1 ）, which secretes anti-lung adenocarcinoma monoclonal antibody, and was transferred into cDNA. Based on the FRl （framework region l） and FR4 conserved regions of LC-1 gene, the variable regions of heavy chain （Vh） and light chain （Vl） were amplified, and the Vh and modified Vl were connected to single chain Fv （ScFv） by SOE-PCR （splice overlap extension PCR）. The modified ScFv was fused with green fluorescent protein （GFP） and introduced into E. coli JM109. The fusion protein induced by lPTG （Isopropylthiogalactoside） was about 57000 on a 10% SDS-PAGE gel （10% Sds Polyacrylamide Gel Electrophoresis）, and primarily manifested as inclusion bodies. The renatured protein purified by Ni-NTA Superflow resins showed ability to bind to antigen on SPC-A-l lung adenocarcinoma. In addition, the induced host cells fluoresced bright green under 395 nm wavelength, which indicated that the expected protein with dual activity was expressed in the prokaryotic system. The ScFv with GFP tag used in this research can be applied as a new reagent to detect immunological dye, and provide a feasible way to detect adenocarcinoma in a clinical setting.
基金support from the National Institutes of Health(Grant R35GM133505)the National Science Foundation(Grant no.2237129)the University of Houston.
文摘The introduction of super-resolution microscopy(SRM)has significantly advanced our understanding of cellular and molecular dynamics,offering a detailed view previously beyond our reach.Implementing SRM in biophysical research,however,presents numerous challenges.This review addresses the crucial aspects of utilizing SRM effectively,from selecting appropriate fluorophores and preparing samples to analyzing complex data sets.We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM.Emphasizing the integration of SRM with other analytical methods,we aim to overcome inherent limitations and expand the scope of biological insights achievable.By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives,we aim to empower researchers to explore complex biological processes with enhanced precision and clarity,thereby advancing the frontiers of biophysical research.
