Biomedical Engineering Reference
In-Depth Information
Dissolve in a centrifugation tube 10 mg wheat germ agglutinin
(WGA) in 5 ml of Soln. A. Add 5 g of wet, ice-cold activated gel
(from BrCN activation; cf. Protocol 3.6.1) and agitate the mixture
atarollershakerfor2h at RT or overnight at 4
◦
C.
Important!
Don't use a magnetic stirrer bar.
After incubation, add 10 ml Soln. B and block at RT further 2 h.
Wash the gel consecutively with 50 ml each of Soln. C and Soln. D
and repeat this step. Finish coupling by washing on a filter funnel
with 100 ml of Soln. E, suck off the buffer and resuspend then in
5 ml of Soln. E. Store the gel in a refrigerator.
The coupling yield is about 60%. If necessary, check the binding
Testing capacity
capacity by adsorption of fetuin and elution with 0.1 M N-acetyl-
glucosamine in PBS.
The immobilization of any other protein to cyanogen bromide-
activated or N-hydroxysuccinimide- (NHS) activated gels is done
the same way.
3.6.2.4 Affinity Purification of HRP
A
PBS
Solutions/Reagents
α
B .1M
-methyl-d-glucoside or -mannoside in PBS
C1M NaCl, 1 mM CaCl
2
,1mMmgCl
2
, 0.02% Thimerosal (w/v)
in 0.1 M acetate buffer, pH 6.0, supplemented with 1 mM
MnCl
2
after adjusting pH
/
Dissolve the crude horseradish peroxidase (HRP) to about 10 mg
ml
in Soln. A and centrifuge.
Wa s h a c o l u m n w i t h 5 ml Concanavalin A bed (1
×
6.4 cm)with
50 ml ofSoln.AatRT.Applyupto20ml oftheaboveHRPsolution.
Whenallofthedarkbrownsolutioniswithinthegel,washwith
Soln.Awithaflowrateofabout1ml
/
min at RT, until the eluate is
colorless. If the capacity of the column is not sufficient, the eluate
may be rechromatographed after regeneration of the column.
Elute the bound colored HRP with Soln. B at 1 ml
/
min.
Regenerate the column with 25 ml of Soln. C and store it in
a refrigerator until reusing.
3.6.2.5 Affinity Chromatography of Immunoglobulins
on Immobilized Antibodies (Immunoaffinity
Chromatography, IAC)
A
PBS
Solutions/Reagents
B .1M glycine-HCl buffer, pH 2.5
C .5M phosphate buffer, pH 7.5, or 0.5 M Tris buffer pH 8.0
BrCN-immobilized IgG
Dilute the respective antiserum, e.g., goat anti-(rabbit IgG) anti-
serum, to about 10 mg
/
ml with Soln. A and centrifuge or filter
µ
through a 0.45-
m membrane filter.
