IF Labeling Protocol

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 200µg of αS preformed fibrils.

Dilute Hu-WT-αS fibrils (PFFs) to a concentration of 1 in PBS followed by sonication (1 Sec ON/1 Sec OFF) for 0h 2m 0s with 20% amplitude.

Mix diluted PFFs with component B (NaHCO3), then added freshly prepared reactive dye (component A) and incubated for 0h 15m 0s at Room temperature .

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 16000x g.

Collect the filtrate as labeled PFFs.