Bio-Rad Affi-Gel Hz Hydrazide Gel - Manual (Page 10)

4. Transfer the gel buffer slurry to a coupling reaction tube. If less
than 5 ml of gel is to be used for coupling, wash only the vol-
ume of gel to be coupled. Unused gel should remain in iso-
propanol and be stored at 4 °C.
4.4B IgG Coupling
As previously recommended, the IgG concentration should be
between 1-5 mg/ml of gel. The total IgG sample volume limitation
of 5 ml is suggested to facilitate buffer exchange and desalting in the
Econo-Pac 10DG columns. Slightly larger sample volumes will
exist at the time of coupling to Affi-Gel Hz gel.
1. Add oxidized, desalted IgG sample to gel in the reaction tube.
Cap securely and rotate end-over-end for 10-24 hours at room
temperature.
2. After coupling reaction is complete, pour gel/IgG slurry into
the 1.0 x 10 cm Econo-Column ® chromatography column pro-
vided. Collect the column eluant and measure the volume.
3. Wash the Affi-Gel Hz immunoaffinity column with 1 column
volume of a suitable buffer containing 0.5 M NaCl (e.g., PBS 0.5
M NaCl, pH 7.0). Collect the column eluant and save for effi-
ciency determination.
4. Wash the column with an application buffer containing 0.02%
sodium azide. If buffers other than PBS 0.5 M NaCl are to be
used, equilibrate the column in 10 volumes of this buffer. Place
yellow end cap securely onto bottom of the Econo-Column
chromatography column. Replace top cap and store column
with buffer above the gel bed at 4 °C until ready to use.
4.4C Calculation of IgG Coupling Efficiency
The efficiency of IgG coupling to Affi-Gel Hz gel can be cal-
culated indirectly. Quantitation of the difference in IgG present
before and after coupling will enable efficiency determination. IgG
coupling can be calculated accurately for samples of high purity.
For samples contaminated with other glycoproteins, the percent-
age of IgG coupled cannot be calculated by total protein determi-
nation.
For Sample of High Purity
Measure the absorbance at 280 nm in a quartz cuvette against
an appropriate buffer blank. Dilute the IgG sample to obtain
absorbance values between 0.1 and 1.0.
8
LIT101C 9/1/98 9:40 AM Page 8