DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 21

NMR requires high magnetic fields and radio-frequency pulses to al-
ter the spin state of nuclei. These spin states differ depending on the
environment the nuclei are in. Thus, the nature of nearby atoms and
their distances influence the chemical shifts of the nuclei that can be
observed by NMR. Only one naturally occurring atom in proteins, hy-
drogen (1H), can be observed by NMR. Therefore, proteins are usually
uniformly labeled with the other atoms that can be observed by NMR,
13-carbon or 15-nitrogen.
Quaternary protein structure:
Protein-protein interactions
Proteins do not always function alone, but frequently function as
dimers, trimers, or higher order polymers of single proteins (homod-
imers, homotrimers, etc.) or more than one protein (heterodimers, het-
erotrimers, etc.). A number of methods have been developed to deter-
mine whether a protein exists in native form or as part of a higher order
polymer within cells.
Non-reducing/non-denaturing gels and gradients
These are often used to isolate proteins from cellular extracts in their
native states. The identities of the component chains can then be iden-
tified by resolving them on denaturing and reducing/denaturing gels to
determine if proteins are non-covalently or covalently associated, as
described in detail earlier in this chapter. This method works well if the
identity of one or more of the interacting proteins is known, and is often
combined with co-immunoprecipitation studies.
Co-immunoprecipitation
Co-immunoprecipitation uses antibodies to one component of a pro-
tien complex to immunoprecipitate it, causing the antibody-bound com-
plex to fall out of solution. If the protein is stably bound to another protein,
both interacting proteins will be immunoprecipitated by the antibody. Al-
ternatively, protein A or protein G coupled to a solid support can be used
to facilitate the isolation of the antibody-bound antigens (see Chapter 4
for more information). The immunoprecipitated material can be dena-
tured in SDS and resolved on SDS-PAGE gels. Interacting proteins can
then be identified by western blotting, if the identity of the interacting
protein is suspected. If unknown proteins are suspected, other meth-
ods are often used. For example, the proteins can first be radioactively