Biomedical Engineering Reference
In-Depth Information
lection of lectins and their specificities is given in Table 2.16. One
should keep in mind that most of the lectins are specific for oligosac-
charides; monosaccharides compete mostly in higher concentra-
tions (Cunnings 1994).
For checking the specificity of interactions between glycopro-
tein and lectin, an incubation in the presence and the absence of 1 -
5 mM of the respective monosaccharide, e.g.,
α
-methylmannoside
for ConA or GlcNAc for WGA, cf. Table 2.16), is recommended.
For identification the lectins must be labeled. The best la-
bels are biotin (streptavidin-enzyme conjugate) or digoxigenin
(anti-digoxigenin antibody-enzyme conjugate), but in the case
of Concanavalin A, direct labeling with HRP is possible (see be-
low).
/
A 0mg
ml of protein (e.g., serum albumin) in 0.1 M sodium
Solutions/Reagents
acetate, pH 4.5 are incubated with periodic acid (final concen-
tration 10 mM)atRTfor6h. Then add glycerol to 10 mM and
dialyze twice against PBS for 2 h each (stock solution). For use
dilute 1:20 with PBS.
or 0.5% polyvinylpyrrolidone (PVP) (w/v) in PBS
or 1% Tween 20 (w/v) in PBS (Attention! This concentration of
Tween 20 sometimes removes proteins form the membrane!)
B1mM CaCl
2
,1mM MnCl
2
in PBS
Block the membrane after electrotransfer with Soln. A for 30 min
at RT. Wash three times with PBS.
Incubate with the biotin or digoxigenin-labeled lectin, diluted
in Soln. A to 5 - 50
µ
/
g
ml,for1h at RT. Wash at least three times
with PBS or TBS.
Incubate with streptavidin-HRP conjugate and anti-digoxigenin
antibody conjugate for 15 - 30 min at RT. Wash again thoroughly.
Stain as described in Protocols 2.5.4 or 2.5.5.
If the lectin Concanavalin A shall be used, dilute it in Soln. B.
Incubateasdescribedabove.Afterwashing,incubatewith50
µ
/
ml
HRPinSoln.Bfor30min, wash with Soln. B again, and stain as
described in Protocols 2.5.4.1 or 2.5.5.1.
g
References
Glass WF II, Briggs RC, Hnilica LS (1981) Anal Biochem 115:219
2.5.7 General Carbohydrate Detection on Western Blots
Carbohydrates are easily oxidized by periodate to aldehydes which
react with primary amines or hydrazines. If the formed hydrazide
carries a specific ligand, e.g., biotin or digoxigenin, these ligands
immobilized via the blotted macromolecule are very sensitively
detected by the respective enzyme conjugates.
