Affinity Targeting Schemes for Biomarker Research - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 199

TEV cleavage
site

B protein of interest

A

TEV cleavage
site

bind to IgG-Fc column

B protein of interest protein of interest

A

cleave with a protease from
tobacco etch virus

B protein of interest protein of interest

bind to calmodulin column

B protein of interest protein of interest

released by the addition of
Ca(II)

B protein of interest

protein A,
= IgG binding
domain

calmodulin
= binding
peptide

B

A

identify proteins by
proteomic methods

other proteins
in complex

FIGURE 4 An illustration of the tandem af nity puri cation method using a protein tagged with calmodulin binding

peptide and protein A.

of proteins and peptides are available from
multiple vendors.
A major issue with avidin:biotin af

protein. The binding af nity of biotin to mono-

meric avidin is much lower than that of tetra-
meric avidin. Elution for monomericavidin/
streptavidin columns can be achieved with
either weak acid or use of biotin as a displacer.
Immobilized avidin/streptavidin matrices are
also useful in immobilizing biotinylated proteins
and small ligands. The fact that avidin/strep-
tavidin binds to biotin with such high af

nity chro-

matography is the dif culty of eluting bio-

tinylated species from native tetrameric avidin
columns. Elution conditions are so harsh that
equipment, columns, and analytes can be
harmed in the process. This problem is
frequently addressed by using a monomeric
avidin or streptavidin column. Monomeric
avidin is generally produced by dissociation of
tetrameric avidin, whereas monomeric streptavi-
din is generally produced as a recombinant

nity
precludes elution of biotinylated proteins from
sorbents. For example, antigens can be dissoci-
ated from avidin:biotin immobilized antibody
without elution of

the antibody from the