Dec 02, 2022
NHS-ester-protein-labeling
- 1University of California, Berkeley
Protocol Citation: Liv Jensen 2022. NHS-ester-protein-labeling. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d2zpg3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 02, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 73494
Keywords: ASAPCRN
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Abstract
Protocol for labeling a purified protein with an NHS ester fluorescent dye.
Materials
-ATTO565 NHS ester (Sigma Cat. #72464)
-Purifiedunlabeled protein
-G-25 desalting column (Cytiva Cat. #28918007)
-Labeling buffer: 50mM HEPES pH8.0, 150mM NaCl, 2mM TCEP
-Quench buffer: 50mM Tris pH 8.0, 150mM NaCl, 2mM TCEP
-Nanodrop spectrophotometer
Mix 40μM unlabeled protein with 80μM ATTO 565 NHS ester dye in labeling buffer.
Incubate 1 hr at room temperature.
Buffer exchange the reaction into quench buffer over a pre-equilibrated G-25 desalting
column.
Assess labeling efficiency by measuring the ratio of absorbance at 280 and 564 nm,
correcting for dye absorbance at 280nm, using a Nanodrop spectrophotometer