Biomedical Engineering Reference
In-Depth Information
loading, wash the column with Soln. B until the UV absorption
at 280 nm is below 0.01. Elute the bound immunoglobulin with
Soln. C. Fractions giving absorption A
1 cm
280
>
0.2 are collected and
immediately neutralized with Soln. D. Centrifuge the combined
eluates to remove aggregates. If necessary, dialyze the eluate three
times against a 20-fold volume of PBS for 2 h each.
Regenerate the column with 60 - 75 ml of Soln. A.
Determine the immunoglobulin concentration of the eluate
photometrically (cf. equation d), Chap. 1.1.7).
Store the protein solution either in the presence of a biocide
as sodium azide or Thimerosal a 4
◦
C or add glycerol to a final
concentration of 30 - 50% (w/v) and store at −20
◦
C.
References
Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring
Harbor Laboratory. p 310
3.6.3 Activation of Sepharose with Epichlorohydrin
Epoxy-activated gels are especially used for immobilization of car-
bohydrates.
A1N NaOH
Solutions/Reagents
Mix 30 g of wet Sepharose CL-4B with 40 ml of Soln. A, 5 ml
epichlorohydrin and 25 ml pure water. Incubate at 40
◦
C with gentle
agitation for 2 h. Then wash on a sintered-glass funnel with 500 ml
water and suck off remaining water. Activated gel is stable at 4
◦
C
for several days.
Caution!
Epichlorohydrin is toxic. Use a hood!
3.6.3.1 Determination of Epoxy Residues
A .3M Na
2
S
2
O
3
(3.226 g Na
2
S
2
O
3
·
5H
2
Oin10ml pure water,
Solutions/Reagents
adjusted to pH 7.5)
B 0mM HCl
Add 3.0 ml of Soln. A to 1.0 g of wet gel. Slurry the mixture with
20 ml ddH
2
Oafter30min atRTandtitratewithSoln.BtopH7.0.
Use a glass pH electrode for monitoring of pH.
One milliliter of used solution is equivalent to 10
µ
Moles of
epoxy groups. After determination of the specific gravity of wet gel
calculate the content.
3.6.4 Immobilization of Monosaccharides (Fucose)
A1N NaOH
Solutions/Reagents
fucose
ethanolamine
