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Modificación química del residuo de triptófano en un dominio recombinante de Ca2+-ATPasa N para el estudio del triptófano-ANS FRET
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Chemical Modification of the Tryptophan Residue in a Recombinant Ca2+-ATPase N-domain for Studying Tryptophan-ANS FRET
DOI:

12:07 min

October 09, 2021

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Chapters

  • 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

Summary

Automatic Translation

ANS se une al dominio N recombinante Ca2+-ATPasa. Los espectros de fluorescencia muestran un patrón similar a FRET sobre la excitación a una longitud de onda de 295 nm. La modificación química del Trp mediada por NBS apaga la fluorescencia del dominio N, lo que conduce a la ausencia de transferencia de energía (FRET) entre el residuo de Trp y el ANS.

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