Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 9)

6. Immediately proceed to desalting, Section 4.3.
4.3 Desalting Procedure
Immediately after the 1 hour oxidation, it is necessary to remove
the sodium periodate from the IgG solution. Sodium periodate
remaining in the IgG sample will adversely affect coupling effi-
ciency. This desalting procedure is the same as that for buffer
exchange (Section 4.1). It is important not to collect and pool frac-
tions beyond the protein peak, since this will result in sodium peri-
odate contamination of the oxidized IgG sample.
1. The Econo-Pac 10DG column(s) used in the buffer exchange
should be washed and equilibrated in diluted coupling buffer, pH
5.5.
2. Follow the buffer exchange procedure in Section 4.1. Remember
to limit the sample volume to 3.0 ml per column, per run. If a
larger sample volume requires multiple desalting runs, wash
each Econo-Pac 10DG column with at least 20 ml diluted cou-
pling buffer after each desalting run.
3. Reserve a small aliquot to determine starting IgG concentra-
tion. This aliquot will be used to calculate IgG coupling effi-
ciency. Measure the volume of oxidized IgG to be coupled.
4.4 Coupling of Oxidized IgG to Affi-Gel Hz
Hydrazide Gel
4.4A Washing Affi-Gel Hz Hydrazide Gel
Affi-Gel Hz hydrazide gel is supplied in isopropanol.
Warning: Isopropanol is poisonous and flammable. Keep away
from heat, sparks, and open flame. May cause eye burns and
skin irritation. Avoid breathing vapor as it irritates eyes, nose,
and throat. Wear gloves and eye protection.
Just prior to coupling, Affi-Gel Hz gel must be washed with
diluted coupling buffer, pH 5.5, to remove isopropanol.
1. With a pipet, transfer the gel/isopropanol slurry to a clear 15
ml tube and allow the gel to settle.
2. Remove isopropanol supernatant, add 10 ml of diluted coupling
buffer, pH 5.5, and mix well. Allow the gel to settle. Repeat.
3. Remove the supernatant above the gel. Add 5 ml of diluted cou-
pling buffer, pH 5.5.
7
LIT101C 9/1/98 9:40 AM Page 7