The most straightforward way to determine interaction partners of a peptide is to use the peptide as bait in affinity pull-down experiments and then by direct detection of binding proteins. Pull-down assays are useful for confirming the existence of protein-protein interaction. The interaction can be identified by other research techniques such as co-immunoprecipitation. The pull-down assay can be used as an initial screening assay for identifying unknown protein-protein interactions. The synthetic peptides are usually used in the verification of postulated protein-protein interactions by competitive disruption of binding.
Biotinylated peptides containing a specific function domain and corresponding control unmodified peptides, can be immobilized onto beads, which are avidin-conjugated. The beads are then incubated with your target samples. These samples can be a nuclear extract, cell lysates, or purified recombinant protein. After washing steps, the unbound proteins are removed. Bound proteins can then be eluted and analyzed by SDS/PAGE. By comparing proteins bound to modified and unmodified peptides, we can identify the specific function of certain protein.
Biotinylated peptides with specific modifications can be chemically synthesized at >80% purity. Peptides should be approximately 15-20 amino acids in length with the modification of interest in the center of the sequence and at least 6-8 flanking residues on each side. Usually, biotin can be labeled at either N terminus or C-terminus. We recommend N terminus modification for its higher success rate. The turnaround time will be shorter. However, the C terminus modification requires an additional Lysine.
Pull-down Protocol of Biotin-labeled Peptide
Biotinylated peptides are conjugated to avidin beads to be used for the peptide pulldown. Immobilized streptavidin beads were loaded with biotinylated peptide before incubation with cell lysates. Cells were lysed in 1% (v/v) NP-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and protease inhibitors. An equal amount of protein was incubated with the respective immobilized peptides at 4 °C for 6 h. After extensive washes, bound proteins were eluted by boiling in SDS sample buffer. The bait peptides with their bound proteins were cleaved off the beads by using 50 mM DTT. Eluates from the active and control bait peptide pull-downs were used for further analysis.
Sketch for streptavidin-biotin bond-force measurements. Because of their exceptionally high binding affinity, two of the most prominent ligand-receptor pairs are streptavidin-biotin and avidin-biotin. Both proteins have a tetrameric structure so that they can bind up to four ligands.
An hsp70 (heat-shock protein of relative molecular mass 70K) can distinguish only unfolded forms of protein. To study the amino acid preferences, Gregory C. Flynn et al. used the random-sequence peptides to fill the binding site of Binding immunoglobulin protein (BiP). It was found that the binding site of BiP shows considerable specificity. The critical residues or Hot spots make a dominant contribution to the free energy of binding. If the key amino acids are mutated, the protein-protein interactions can be disrupted.
Reference: Gregory C. Flynn, Jan Pohl, Mark T. Flocco & James E. Rothman. Peptide-binding specificity of the molecular chaperone BiP. 24 October 1991, Nature 353, 726-730 doi:10.1038/353726a0.
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