Biomedical Engineering Reference
In-Depth Information
Apply 1 ml of the clear solution on top of a column containing
the immobilized antigen, e.g., rabbit IgG. The bed size depends on
the capacity of the support; in this example it should be 4 - 5 ml.
Wash with Soln. A until the O.D.
280
of the eluate is
<
0.05.
Pipet 0.2 ml of Soln. C into tubes for fraction collection. If chro-
matography is performed in the cold, the phosphate buffer Soln. C
tends to crystallize; therefore, add phosphate buffer immediately
before use.
Elute the bound immunoglobulin with Soln. C at a flow rate of
1 ml
/
min. Mix the eluate thoroughly with the buffer within the col-
lector tubes and monitor the elution by measuring UV absorption
at 280 nm.
Combine fractions with A
280
>
0.4, dialyze against the 100-fold
volume PBS for at least 2 h, and centrifuge with 2000
g for 20 min.
If necessary, concentrate the dialysate by ultrafiltration.
A milder elution is possible using 2 MMgCl
2
, pH 5.0 instead
of Soln. B. In this case Soln. C is omitted. For other conditions of
elution see Table 3.7.
Especially if samples with a high content of unspecific proteins,
e.g., sera, are processed by immunoaffinity chromatography, it is
recommended to use a sequential washing/elution protocol: When
allsampleisapplied,washthecolumnwithPBSorTBSuntilO.D.
280
<
0.1, then with 1 column volume 1 Msodium chloride, followed by
5 vol. PBS or TBS. Elute with 1 vol. elution buffer, e.g., glycine-HCl
pH 2.5, then apply 1 vol. alkaline buffer, e.g., 0.1 M borate pH 10.
Finally, regenerate the column with PBS or TBS.
×
References
Jack GW (1994) Molec Biotechnol 1:59
3.6.2.6 Affinity Chromatography of Rabbit IgG
on Protein-A Supports
The specificity of protein A for immunoglobulins of different
species and subclasses is given in Table 4.6.
A
PBS, pH 7.8
Solutions/Reagents
B .5M NaCl, 50 mM sodium phosphate, pH 7.8
C .1M sodium citrate, pH 3.0
3
D1N NaOH
RinseaproteinAcolumn(4-5ml bed volume; 15
×
22.5 and 15
×
/
28.5 mm, respectively) with 40 - 50 ml of Soln. A, flow rate 1 ml
min.
Dilute 2 - 5 ml of rabbit antiserum with the same volume of Soln. A
and apply with the same flow rate to the column. After sample
3
Since the immunoglobulins purified by this method are mostly used
for immobilization, buffers containing NH
2
groups,asTrisorglycine,
should be avoided.
