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Immunoaffinity Chromatography 57 A sample chromatogram for an immunoaffinity purification is shown in Fig. 1. In this figure, the large flow-through peak can be seen. This flowthrough peak is comprised of contaminating proteins, DNA, and other molecules cleared by the immunoaffinity chromatography. The quality of the eluate can be judged from the sodium dodecyl sulfate–polyacrylamide gel electrophoresis mAU A Loading Washing Elution 1500 1000 500 Load PBS pH 2.24 line wash pH 2.24 elution PBS line wash PBS column wash 0.05% NaN3 PBS, 0 0 50 100 150 200 250 300 ml B mAU 80 60 40 20 Load PBS line wash pH 2.24 elution pH 2.24 Wash PBS Line PBS column wash 0.05% NaN3 PBS, 0 0 50 100 150 200 250 300 ml Fig. 1. Representative A 280 nm chromatograms of immunoaffinity chromatography at two magnifications. (A) At lower magnification, the relative clearance of various contaminants can be seen by comparing the flow-through peak to the elution peak. (B) At higher magnification, the elution peak can be seen to be quite sharp. It is preceded by a low flat peak that has no significance; this section of the chromatogram was generated during line flushing of the chromatography system.
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Immunoaffinity Chromatography 57<br />
A sample chromatogram for an immunoaffinity purification is shown in<br />
Fig. 1. In this figure, the large flow-through peak can be seen. This flowthrough<br />
peak is comprised of contaminating proteins, DNA, and other molecules<br />
cleared by the immunoaffinity chromatography. The quality of the eluate can<br />
be judged from the sodium dodecyl sulfate–polyacrylamide gel electrophoresis<br />
mAU<br />
A<br />
Loading<br />
Washing<br />
Elution<br />
1500<br />
1000<br />
500<br />
Load<br />
PBS<br />
pH 2.24 line wash<br />
pH 2.24 elution<br />
PBS line wash<br />
PBS column wash<br />
0.05% NaN3<br />
PBS,<br />
0<br />
0 50<br />
100 150 200 250 300 ml<br />
B<br />
mAU<br />
80<br />
60<br />
40<br />
20<br />
Load<br />
PBS<br />
line wash<br />
pH 2.24<br />
elution<br />
pH 2.24<br />
Wash<br />
PBS Line<br />
PBS column wash<br />
0.05% NaN3<br />
PBS,<br />
0<br />
0 50 100 150 200 250 300 ml<br />
Fig. 1. Representative A 280 nm chromatograms of immunoaffinity chromatography<br />
at two magnifications. (A) At lower magnification, the relative clearance of various<br />
contaminants can be seen by comparing the flow-through peak to the elution peak.<br />
(B) At higher magnification, the elution peak can be seen to be quite sharp. It is<br />
preceded by a low flat peak that has no significance; this section of the chromatogram<br />
was generated during line flushing of the chromatography system.
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