Protein Protocols Protein Protocols

890 Fotinopoulou and Turner
Fig. 4. MAA binding before (solid line) and after (dotted line) neuraminidase treatment
(buffer plot subtracted from both sensograms).
Table 1
Typical Results for Different Glycoproteins with Different Lectins
ConA SNA b SNA a MAA b MAA a PNA b PNA a DSA AAA
IgG 1680 1344 50 125 26 24 123 133 71
Fetuin 1776 1230 78 624 15 15 180 230 32
Rec1 2768 1122 20 110 25 26 130 111 22
Rec2 1203 1110 25 325 22 14 297 142 15
b,a Before and after neuraminidase treatment. IgG and fetuin were commercial products, while rec1
and rec2 are recombinant glycoproteins. Buffer readings gave values of 20–35 RU. See Note 8.
1 unit pH below pI, for pI 3.5–5.5, use 0.5 pH units below pI. For pH range of 4–5.5
10 mM acetate buffer is recommended.
2. The time of activation may be modified to regulate the amount of ligand immobilized.
3. For a given pH the ideal ligand concentration is the lowest value that gives maximum
preconcentration. Usually, a suitable concentration will be in the range 10–200 µg/mL. A
target response is a level of immobilization that gives about 0.07 pmol ligand/mm 2 . That
corresponds to mol wt/15 RU.
4. Analyte concentration can be chosen between 5 µg/mL and 500 µg/mL.
5. A reading value above 30–40 RU is considered as real binding.
6. After regeneration the baseline should be at the same level as before lectin binding. A
drift in the baseline may appear after repeated regeneration, which may be due to loss of
Neu5Ac.
7. Different enzyme incubation times may apply depending on the enzyme properties.
8. Table 1 gives typical results for different glycoproteins with different lectins. The following
conclusions can be drawn from these results: IgG has N-linked chains (ConA),
α2–6 Neu5NAc (SNA) and fucose (AAA) and it does not have any O-linked chains (PNA)
or any α2–3 Neu5NAc (MAA); Fetuin has N- and O-linked chains, α2–6 Neu5NAc and