What experimental techniques can be used to identify binding partners for a given protein? I know that if you have some candidates that may bind to a given protein, then you can use techniques such as pull-down or coimmunoprecipitation to determine whether the protein binds to those putative binding partners. But can you screen for its binding partners when basically there is no knowledge on what the given protein might bind to?
(1) obtain antibody of said PROTEIN (search catalog or custom-made).
(2) pull down said PROTEIN with antibody and techniques you mentioned.
(3) identify other proteins in the pulled-down complex with following method:
(From here) There are two main methods of amino acid sequencing: mass spectrometry and Edman degradation with a protein sequenator.
Automated Edman amino acid sequencers are offer convenient analysis of polypeptides of up to 50 amino acids long. This process is generally characterized by seven steps:
- Break apart disulphide bridges in the protein with a reducing agent
- Separate the protein complex and purify the chain(s)
- Determine the amino acid composition and terminal AAs per chain
- Fragment each polypeptide chain
- Recreate the AA sequence using these fragments
- Repeat with different fragment patterns to mitigate errors
Identification via mass spectrometry is increasingly preferred as it overcomes many of the established limitations of Edman degradation. But there are various techniques within protein mass spectrometry that make amino acid sequencing via MS techniques harder to define in brief.