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組換えCa2+-ATPaseNドメインにおけるトリプトファン残基の化学修飾
JoVE Journal
Biyokimya
Author Produced
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JoVE Journal Biyokimya
Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET

組換えCa2+-ATPaseNドメインにおけるトリプトファン残基の化学修飾

DOI:

12:07 min

October 09, 2021

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Bölümler

  • 00:03Introduction
  • 01:15Determination (in silico) of the ANS and SERCA N‐Domain Interaction
  • 03:09Expression and Purification of the Recombinant N‐Domain
  • 03:39Monitor the Formation of the ANS‐N‐Domain Complex Based on ANS and N‐Domain Fluorescence Intensity Changes
  • 07:38N‐Domain Intrinsic Fluorescence Titration by Trp Chemical Modification with NBS
  • 08:43Titrate the NBS Modified N‐Domain with ANS by Recording Fluorescence Spectra at 25°C
  • 09:37Evidence of ANS Binding to the Chemically Modified N‐Domain by Excitation at λ = 370 nm
  • 10:32Results Overview
  • 11:30Conclusions

Özet

Otomatik Çeviri

ANSはCa2+-ATPase組換えNドメインに結合する。蛍光スペクトルは、295nmの波長で励起時にFRET様パターンを表示します。TrpのNBS媒介化学修飾は、Nドメインの蛍光を発し、Trp残基とANSとの間のエネルギー移動(FRET)の欠如をもたらす。

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