Bio-Rad DEAE Affi-Gel Blue Gel - Manual (Page 7)

Application Buffers
Buffer ionic strength is a critical parameter in DEAE Affi-Gel blue chro-
matography. Small variations in salt concentration and pH significantly effect
sample purity and/or sample recovery. The appropriate buffer composition for
human and rabbit serum chromatography has been determined in our laborato-
ries as follows:
Serum Buffer
Rabbit 0.02 M Tris-HCl, pH 8.0, containing 0.028 M NaCl
Human 0.02 M K
2
HPO
4
, pH 8.0
The Econo-Pac serum IgG purification columns can also be used to purify
serum IgG from other species. For species such as goat, rat, or sheep, the rab-
bit buffer is suitable.
Regeneration buffer
The columns should be regenerated after each chromatography run. This will
insure removal of any bound proteins an prevent cross-over contamination from
one run to the next. Suitable regeneration buffers include 1.5 M sodium thio-
cyanate or 2 M guanidine HCl in the application buffer.
2.3 Sample Preparation
Bio-Rad’s Econo-Pac 10DG desalting columns are recommended for serum
or ascites preparation. Each desalting column can prepare up to 3 milliliters of
serum. The serum sample is then collected in 4 milliliters of application buffer,
so sample dilution is minimal. The whole procedure takes approximately 30
minutes. Alternatively, dialysis of the serum sample against the appropriate
application buffer may be used before chromatography to reduce the serum salt
content and adjust the pH.
2.4 Standard Serum IgG Purification Procedure
1. Discard the buffer above the top frit of an Econo-Pac serum IgG purifica-
tion column and snap off the bottom tip.
2. Prewash the first time use only: Wash the column with 40 ml of prewash
buffer (fill the column twice), followed by 40 ml of the appropriate appli-
cation buffer (human or rabbit). Allow the buffer to drain to the top of the
frit. The column will not run dry. Proceed to step 4.
[If the column has already been prewashed, begin the procedure with step 3.]
3. Equilibrate the column with 30 ml of application buffer.
4. Apply prepared sample to the column. See label on column package for
exact column serum capacity.
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