**Protocols
of FAC-FD**

__Principle
of FAC-FD__

The analytical
principle is literally based on quantitative affinity chromatography. An excess
volume of glycan-containing solution is applied onto the lectin-immobilized
column. If the glycan binds to the immobilized lectin, the elution front volume
(*V*) is delayed compared to the negative control glycan (*V*_{0})
(Fig. 2).

The *V*-*V*_{0}
value corresponds to the binding affinity between the glycan and the
immobilized lectin. From the obtained *V*-*V*_{0} and *B*_{t}
(the effective lectin content expressed in mol), *K*_{d} is
calculated from the following formula:

[A]_{0} (*V*-*V*_{0})
= *B*_{t} [A]_{0} / ([A]_{0} + *K*_{d})
(equation 1)

,where [A]_{0}
is the initial glycan concentration in M. Since [A]_{0} (~5.0 x 10^{-9}
M) required for FAC-FD analysis is negligibly small compared with the *K*_{d}
(10^{-3}-10^{-7} M) of general sugar-protein interactions, the
following formula can be derived from equation 1:

*K*_{d} = *B*_{t}
/ (*V*-*V*_{0}), if *K*_{d }>>[A]_{0}
(equation 2)

,where the affinity
constant (*K*_{a}) is the inverse of *K*_{d} (*K*_{a}
=1/ *K*_{d}).

__Analytical
scheme__

FAC-FD analysis is
performed using the automated FAC-FD system in a highly systematic manner. The analytical
scheme is divided into four steps: (*i*) preparation of lectin-immobilized
columns, (*ii*) 1^{st} screening, (*iii*) 2^{nd}
screening, and (*iv*) concentration-dependence analysis.

*Preparation of
lectin-immobilized columns*: In most cases, lectins are immobilized on *N*-hydroxysuccinimide
(NHS)-activated Sepharose 4FF using amine coupling chemistry.

*1 ^{st}
screening using 49 selected PA-glycans*: To validate the
prepared lectin column, binding to 49 PA-glycans, representing glycan
structures derived from various glycoconjugates, is examined as the first
screening. If little or no binding is observed for the first 49 glycans,
another column, immobilizing a still higher concentration of the same lectin, is
prepared. An example of GSL-II isolectin is shown in Fig. 3.

*2 ^{nd}
screening using a full panel of PA-glycans*: If sufficient binding
is observed for a specific group of glycan structures, the second screening
using a full panel of PA-glycans for more detailed and precise analysis is
carried out. In particular cases, the third step of extended analysis is
optionally performed using non-standard or specially elaborated glycans.

*Concentration-dependence
analysis*:
Finally, the effective ligand content (*B*_{t}) value of the
immobilized lectin is determined by a so-called concentration-dependence
analysis followed by a Woolf-Hofstee-type plot for determination of *K*_{d}
as well as *B*_{t} using an appropriate positive saccharide
derivative. In this DB, data are shown in an actual measurement (*V*-*V*_{0})
as well as an association constant (*K*_{a}).