Sulfo-NHS-LC-LC-Biotin (Biotin-XX-SSE) is used for efficient and irreversible biotin labeling of antibodies, proteins and any other macromolecules containing a primary amine group (-NH2), thus permitting the use of streptavidin or affinity couplings for the detection and isolation of proteins; since the negatively charged reagent does not penetrate the cellular membrane, it is only labeled on the cellular surface for labeling.

Biotinylation of exosomal proteins
I. Solution preparation
1. Mother solution preparation: Dissolve Sulfo-NHS-LC-LC-Biotin in DMSO to prepare a 10-100mM mother solution.
2. Working solution preparation: Use water/PBS or other solvents to dilute the Sulfo-NHS-LC-LC-Biotin mother solution to the working solution concentration used in the experiment.
Notes:
1. The mother solution needs to be stored at -20℃ or -80℃ to avoid repeated freezing and thawing.
2. Please dilute the mother solution to the required concentration according to your experimental purpose.
II. Operation steps
1. Mix 200 μg of intact exosomes with 10 mM Sulfo-NHS-LC-LC-Biotin at room temperature for 30 minutes.
2. Four conditions were considered in this experiment:
1) Use excess Sulfo-NHS-LC-LC-Biotin to support complete saturation of exposed lysine residues and potential N-termini.
2) The sulfonate group present in Sulfo-NHS-LC-LC-Biotin prevents the reagent from penetrating the exosome membrane.
3) Sulfo-NHS-LC-LC-Biotin has a 30.5 angstrom spacer arm that improves biotinylation of proteins in their native conformation.
4) Amino acids labeled with Sulfo-NHS-LC-LC-Biotin will add a mass of 452 Da.
After incubation, excess Sulfo-NHS-LC-LC-Biotin can be removed using a 10 KDa MWCO filter. [2]