LifeTein synthesized multiple peptides for the pulldown experiments. The peptides were synthesized with biotin on the N terminus and an aminohexanoic acid linker. Two types of peptides were designed: peptide without acetylation and peptide with multiple lysines (up to five sites) were acetylated in the synthetic peptides.
Pulldown Experiments With LiteTein Peptides
These peptides were bound to streptavidin-agarose beads and used for pulldown experiments using cell lysates. Peptides, streptavidin agarose beads, and cell lysates were permitted to bind for overnight at 4 °C, then beads were pelleted, washed five times using spin columns, and proteins were eluted in sample buffer and analyzed by SDS-PAGE and immunoblotting.
All synthetic peptides had an N-terminal biotin. After incubation, peptides were spotted on nitrocellulose membranes and immunoblotted to detect acetylated Lysine (AcK) or total biotinylated peptide. Data is shown below.
Peptides can be used for ELISA assay. A peptide-based indirect ELISA was used to screen a population of 40 Multiple sclerosis patients and 39 healthy controls. The encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35–55 synthetic peptides were synthesized by LifeTein.
LifeTein’s Synthetic Peptides for ELISA
All MOG peptides or Mycobacterium avium subspecies paratuberculosis (MAP) peptides were synthesized at >90% purity by LifeTein to make sure the ELISA results are clean and consistent. The plates were coated with peptides. After overnight incubation, the plate was blocked, rinsed, and late react with antibodies according to the protocol. In silico analysis identified two peptides belonging to MAP and BCG, which share sequence homology with MOG(35–55). The peptide-based indirect ELISA data showed that sharing of highly conserved linear amino acidic sequences is necessary to elicit antibody-mediated cross-reactivity.
These findings concluded that the presence of MOG (35–55)-specific antibodies in multiple sclerosis pathogenesis. This can be used as a diagnostic biomarker in multiple sclerosis.
… All peptides were synthesized at N90% purity commercially (LifeTein, South Plainfield, NJ 07080 US). Purified peptides were prepared as [10 mM] stock solutions, and were stored in single- use aliquots at −80 °C. 2.3. ELISA …
