Binds and sequesters Ca²⁺ ions. EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid),

So that is mostly likely going to destroy any interactions with the tagged protein.

This is a fairly hydrophilic sequence ((DYKDDDDK)) that a number of very specific antibodied that have been raised to it. Being so hydrophilic, it tends not to denature proteins to which it is fused (presumably hydrophobic peptides could interfere with proteins' folding - the "molten globule" - as the protein is synthesised from the ribosome..

Sm proteins are a faimily that binds small nucleae RNAs, part of the spliceosome.

How does Mass spec allow you to identify the other proteins purified:

Why is this important? What artefacts could occur if you overexpressed the protein? How can you ensure that the protein is expressed at its natural level>

Think of a complex between A , B and C, and D. B and C binds to A, and D only binds B and C when they are bound to A. If we only know about A, and over express it, then it is most likely that B and C will end up on separate molecules of A. So then B and C are never on the same molecule of A, and so D can never bind them. Therefore by over-expressing A you will never find D.

Think of a complex between A , B and C, and D. B and C binds to A, and D only binds B and C when they are bound to A. If we only know about A, and over express it, then it is most likely that B and C will end up on separate molecules of A. So then B and C are never on the same molcule of A, and so D can never bind them. Therefore by over-expressing A you will never find D.

Tryptic digest of the bands found after affinity purification followed by mass spec' to identify the peptide lengths is enough to provide a unique fingerprint that will uniquely match one protein in the genomes (assuming that the genome has been sequenced). By this means you can identify all unknown proteins,.