![단분자 결합 스크린닝. (a) Single-molecule binding assay. (a) TIFR image of surface-immobilized KPNA with control or irradiated EGFR at different times. Scale bar: 10 μm. (b) Number of fluorescent binding spots of 200 nM control or irradiated EGFR with 100nM of immobilized KPNA or KPNB1. (c) Number of binding spots of 200 nM irradiated EGFR and 100 nM immobilized KPNA. The kobs= kon [irradiated EGFR] – koff= 0.21±03 min-1nM-1. (d) Number of fluorescent spots after incubating 200 nM irradiated EGFR with 100 nM KPNA for 1 h and rinsing with T50 buffer. The off-rate constant is obtained during treating the imaging buffer as koff= 0.03±01 min-1. (e) Number of binding spots after incubating different concentrations of irradiated EGFR with immobilized KPNA for 20 min. Km is estimated as 69±16 nM by Michaelis-Menten equation [y = Vmax* x / (Km+ x)]. All binding spot counts were averaged over an imaging area of 2500 μm2](https://nrms.kisti.re.kr/bitextimages/TRKO202200018270/TRKO202200018270_8_image_15.png "단분자 결합 스크린닝. (a) Single-molecule binding assay. (a) TIFR image of surface-immobilized KPNA with control or irradiated EGFR at different times. Scale bar: 10 μm. (b) Number of fluorescent binding spots of 200 nM control or irradiated EGFR with 100nM of immobilized KPNA or KPNB1. (c) Number of binding spots of 200 nM irradiated EGFR and 100 nM immobilized KPNA. The kobs= kon [irradiated EGFR] – koff= 0.21±03 min-1nM-1. (d) Number of fluorescent spots after incubating 200 nM irradiated EGFR with 100 nM KPNA for 1 h and rinsing with T50 buffer. The off-rate constant is obtained during treating the imaging buffer as koff= 0.03±01 min-1. (e) Number of binding spots after incubating different concentrations of irradiated EGFR with immobilized KPNA for 20 min. Km is estimated as 69±16 nM by Michaelis-Menten equation [y = Vmax* x / (Km+ x)]. All binding spot counts were averaged over an imaging area of 2500 μm2")
![(a) Number of fluorescent binding spots between 5 nM of phosphorylated peptide at threonine residue (TAMRA-RRRHINRKRpTLRR, pT-peptide) to 100 nM of immobilized KPNA. The on-rate constant was kon= 0.18±04 min-1. (d) Number of binding spots after incubating 5nM of pT-peptide with 100nM KPNA for 1h and rinsing with T50 buffer. The counts revealed an off-rate constant koff= 0.03±008 min-1. © Number of binding spots after incubating different concentrations of pT-peptide with KPNA for 20min, and Km is estimated to be 6±01 nM from Hill equation[y = Vmax*xn/(kn+xn)] and n=2.33±0.47. All binding spot counts were averaged over an imaging area of 2500 μm2](https://nrms.kisti.re.kr/bitextimages/TRKO202200018270/TRKO202200018270_9_image_1.png "(a) Number of fluorescent binding spots between 5 nM of phosphorylated peptide at threonine residue (TAMRA-RRRHINRKRpTLRR, pT-peptide) to 100 nM of immobilized KPNA. The on-rate constant was kon= 0.18±04 min-1. (d) Number of binding spots after incubating 5nM of pT-peptide with 100nM KPNA for 1h and rinsing with T50 buffer. The counts revealed an off-rate constant koff= 0.03±008 min-1. © Number of binding spots after incubating different concentrations of pT-peptide with KPNA for 20min, and Km is estimated to be 6±01 nM from Hill equation[y = Vmax*xn/(kn+xn)] and n=2.33±0.47. All binding spot counts were averaged over an imaging area of 2500 μm2")
![(a) Number of fluorescent binding spots between 5nM of phosphorylated peptide at threonine residue (TAMRA-RRRHINRKRpTLRR, pT-peptide) to 100nM of immobilized KPNA. The on-rate constant was kon= 0.18±04 min-1. (d) Number of binding spots after incubating 5nM of pT-peptide with 100nM KPNA for 1h and rinsing with T50 buffer. The counts revealed an off-rate constant koff= 0.03±008 min-1. © Number of binding spots after incubating different concentrations of pT-peptide with KPNA for 20min, and Km is estimated to be 6±01 nM from Hill equation[y = Vmax*xn/(kn+xn)] and n=2.33±0.47. All binding spot counts were averaged over an imaging area of 2500 μm2](https://nrms.kisti.re.kr/bitextimages/TRKO202200018270/TRKO202200018270_10_image_1.png "(a) Number of fluorescent binding spots between 5nM of phosphorylated peptide at threonine residue (TAMRA-RRRHINRKRpTLRR, pT-peptide) to 100nM of immobilized KPNA. The on-rate constant was kon= 0.18±04 min-1. (d) Number of binding spots after incubating 5nM of pT-peptide with 100nM KPNA for 1h and rinsing with T50 buffer. The counts revealed an off-rate constant koff= 0.03±008 min-1. © Number of binding spots after incubating different concentrations of pT-peptide with KPNA for 20min, and Km is estimated to be 6±01 nM from Hill equation[y = Vmax*xn/(kn+xn)] and n=2.33±0.47. All binding spot counts were averaged over an imaging area of 2500 μm2")
![Single-molecule pull-down assay. (A) The apparent association rate constant, kobs was obtained by counting the number of NUPR1-bound KPNB1 spots over time upon the addition of 5 nM KPNB1. The association rate constant, kon can be obtained from kobs = kon [KPNB1] - koff which is 0.046 min-1 nM-1. (B) The dissociation rate constant, koff was obtained as 0.018 min-1 by counting the number of NUPR1-bound KPNB1 spots over time right after removing the freely diffusing KPNB1 proteins in the solution by substituting the solution with an KPNB1 free imaging buffer](https://nrms.kisti.re.kr/bitextimages/TRKO202200018270/TRKO202200018270_11_image_16.png "Single-molecule pull-down assay. (A) The apparent association rate constant, kobs was obtained by counting the number of NUPR1-bound KPNB1 spots over time upon the addition of 5 nM KPNB1. The association rate constant, kon can be obtained from kobs = kon [KPNB1] - koff which is 0.046 min-1 nM-1. (B) The dissociation rate constant, koff was obtained as 0.018 min-1 by counting the number of NUPR1-bound KPNB1 spots over time right after removing the freely diffusing KPNB1 proteins in the solution by substituting the solution with an KPNB1 free imaging buffer")
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| 주관연구기관 | 중앙대학교 Chung Ang University |
|---|---|
| 연구책임자 | 함승욱 |
| 보고서유형 | 최종보고서 |
| 발행국가 | 대한민국 |
| 언어 | 한국어 |
| 발행년월 | 2021-06 |
| 과제시작연도 | 2020 |
| 주관부처 | 교육부 Ministry of Education |
| 연구관리전문기관 | 한국연구재단 National Research Foundation of Korea |
| 등록번호 | TRKO202200018270 |
| 과제고유번호 | 1345320008 |
| 사업명 | 개인기초연구(교육부)(R&D) |
| DB 구축일자 | 2023-02-22 |
| 키워드 | 방사선 조사.단백질 운송.항암제.Irradiation.EGFR.Transport protein.anticancer drug. |
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