Jun 25, 2024
IF Labeling Protocol
- Scott Vermilyea1
- 1University of Minnesota, Team Lee
Protocol Citation: Scott Vermilyea 2024. IF Labeling Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q6rql1y/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: June 07, 2024
Last Modified: June 25, 2024
Protocol Integer ID: 102391
Keywords: Protein Labelling, Tetrafluorophenyl, Conjugation
Funders Acknowledgement:
ASAP
Grant ID: 000592
Abstract
This protocol details the Labelling of Immunofluorescent Protein.
Materials
- Alexa Fluor™ 488 Microscale Protein Labeling KitThermo FisherCatalog #A30006
Immunofluorescent Protein Labeling Protocol
Immunofluorescent Protein Labeling Protocol
17m 15s
Alex Fluor 488 Microscale Protein Labelling Kit (Invitrogen, A30006).
According to manufacturer protocol, AF 488 reactive dye has Tetrafluorophenyl (TFP) ester moiety that is more stable in solution than the commonly used succinimidyl (NHS) ester. TFP ester react efficiently with primary amines of protein to form a stable dye-protein conjugate and is independent of pH between 4 and 10.
Single kit can label a total of 200 µg of αS preformed fibrils.
Dilute Hu-WT-αS fibrils (PFFs) to a concentration of 1 undetermined in PBS followed by sonication (1 Sec ON/1 Sec OFF) for 00:02:00 with 20% amplitude.
2m
Mix diluted PFFs with component B (NaHCO3), then added freshly prepared reactive dye (component A) and incubated for 00:15:00 at Room temperature .
15m
Calculate amount of reactive dye using dye/protein molar ratio of 9.
Remove excess dye by resin gel (component E) purification.
Place conjugated reaction mixture of PFFs and reactive dye on the center of the resin bed surface, centrifuge at 16000 x g, 00:00:15 .
15s
Collect the filtrate as labeled PFFs.