Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 7)

in affinity chromatography.
4.1A Dilution of Affi-Gel Hz 10x Coupling Buffer
1. Dilute Affi-Gel Hz 10x coupling buffer 1:10 with distilled,
deionized water and mix well.
2. Check the pH of the diluted coupling buffer with a pH meter. The
pH of the diluted buffer should be 5.5. If it is necessary to cor-
rect the pH of the diluted Affi-Gel Hz coupling buffer, use
1.0 M acetic acid or 1.0 M NaOH to bring the pH to 5.5.
Sodium azide, at a concentration of 0.02%, can be added to the
diluted coupling buffer for long-term storage. However, it is
best to dilute only the amount of 10x concentrate required.
4.1B Buffer Exchange with Econo-Pac 10DG Desalting
Columns
Buffer exchange with Econo-Pac 10DG columns gives a min-
imum dilution of the purified antibody. Sample volumes may vary,
but only 3.0 ml should be run at a time. These columns can be
regenerated and reused if adequately washed after protein collection.
1. Remove the upper cap from the Econo-Pac 10DG column and
pour off the excess buffer above the top frit.
2. Add 20 ml of the diluted Hz coupling buffer, pH 5.5 (fill to the
30 ml mark), and snap off the bottom tip to start the column
flowing.
3. Allow the buffer to drain to the top frit. The column will not run
dry. Flow will stop when the buffer level reaches the top frit.
4. Add up to 3.0 ml of purified IgG sample to the Econo-Pac 10DG
column. Allow the sample to run completely into the column.
If applying a sample of less than 3.0 ml, add the difference in
diluted coupling buffer to the column, allowing it to run com-
pletely into the column (3.0 ml sample volume). Discard the
first 3.0 ml eluted from the column.
5. Add pH 5.5 coupling buffer (120% of the starting sample vol-
ume) to the top of the Econo-Pac 10DG column and collect 0.5
ml fractions. Monitor the absorbance of the fractions at 280
nm. Pool the fractions containing the purified antibody in Affi-
Gel Hz coupling buffer.
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