Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 8)

6. The Econo-Pac 10DG column should be washed with at least 20
ml of diluted coupling buffer, pH 5.5, to regenerate for desalt-
ing use after oxidation.
7. Place the yellow end cap over the column tip snugly to prevent
leakage. Add 5 ml of diluted coupling buffer with 0.02% sodi-
um azide to the column and replace the top cap for column stor-
age.
8. The antibody is now ready for sodium periodate oxidation.
4.2 Oxidation of IgG
The oxidation of purified IgG will require the use of sodium
m-periodate (NaIO
4
).
Warning: Sodium periodate is a powerful oxidant. Avoid con-
tact and inhalation. May be harmful if swallowed. May react
violently with reducing agents, hydrides, and finely powdered
metals. Use only as directed in this manual. Wear gloves and eye
protection.
Affi-Gel Hz oxidizer is used as a stock solution and added to IgG
as required. Since sample volumes will vary, it is necessary that the
ratio of stock solution to sample be given, rather than actual volumes.
Sodium periodate is light sensitive; therefore, oxidation must be
performed in the dark. Maximum storage of the diluted sodium
periodate stock solution is 1 week at 4 °C, in the dark glass vial
provided. At the recommended sodium periodate concentrations,
oxidation of carbohydrate moieties is specific and does not alter
IgG activity.
1. Add 1.2 ml of distilled, deionized water to the sodium periodate
vial (25 mg), replace stopper, and vortex until dissolved.
2. Place the purified IgG sample (in diluted coupling buffer) in a
screw cap, polypropylene, or polystyrene tube. Make certain
that the tube selected will not leak.
3. Add the sodium periodate stock solution at one-tenth the volume
of purified IgG sample (e.g., 400 µ1 of NaIO
4
stock solution to
4.0 ml of purified IgG). For rabbit IgG, add the sodium perio-
date at three-tenths the volume of purified IgG (e.g., 1.2 ml
NaIO
4
to 4.0 ml purified IgG).
4. Secure cap and cover the tube with foil.
5. Rotate the antibody/sodium periodate mixture end-over-end for
l hour at room temperature.
6
LIT101C 9/1/98 9:40 AM Page 6