Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
Affinity Chromatography - Department of Molecular and Cellular ...
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Monoclonal IgM from hybridoma cell culture<br />
HiTrap IgM Purification HP<br />
The technique described here is optimized for purification <strong>of</strong> monoclonal IgM from<br />
hybridoma cell culture, but it can be used as a starting point to determine the binding <strong>and</strong><br />
elution conditions required for other IgM preparations.<br />
Purification option<br />
Binding capacity Maximum Comments<br />
operating flow<br />
HiTrap IgM Purification HP Human IgM, 5 mg/column 4 ml/min Purification <strong>of</strong> monoclonal <strong>and</strong><br />
human IgM.<br />
Prepacked 1 ml column.<br />
HiTrap IgM Purification HP columns are packed with a thiophilic adsorption medium<br />
(2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the<br />
protein <strong>and</strong> the lig<strong>and</strong> has been suggested to result from the combined electron donating<strong>and</strong><br />
accepting-action <strong>of</strong> the lig<strong>and</strong> in a mixed mode hydrophilic-hydrophobic interaction.<br />
Purification example<br />
Figure 19 shows results from the purification <strong>of</strong> monoclonal a-Shigella IgM from<br />
hybridoma cell culture supernatant. SDS-PAGE analysis demonstrates a purity level <strong>of</strong> over<br />
80%. Results from an ELISA (not shown) indicated a high activity <strong>of</strong> the antibody in the<br />
purified fraction.<br />
A 280 nm<br />
mS/cm<br />
2.5<br />
Sample: 75 ml <strong>of</strong> cell culture supernatent<br />
containing a-Shigella IgM, filtered through<br />
a 0.45 µm filter<br />
Column: HiTrap IgM Purification HP, 1 ml<br />
Binding buffer: 20 mM sodium phosphate buffer,<br />
0.5 M potassium sulphate, pH 7.5<br />
Elution buffer: 20 mM sodium phosphate buffer, pH 7.5<br />
Cleaning buffer: 20 mM sodium phosphate buffer, pH 7.5,<br />
30% isopropanol<br />
Flow:<br />
1 ml/min<br />
1.5<br />
0.5<br />
Flow<br />
through<br />
material<br />
Elution<br />
buffer<br />
IgM<br />
Cleaning<br />
buffer<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0 80 100<br />
0<br />
ml<br />
Fig. 19a. Purification <strong>of</strong> a-Shigella IgM on HiTrap IgM Purification HP.<br />
Samples reduced with<br />
2-mercaptoethanol<br />
M r<br />
97 000<br />
66 000<br />
45 000<br />
30 000<br />
20 100<br />
14 400<br />
1 2 3 4 5 6 7 8<br />
Non-reduced samples<br />
M r<br />
97 000<br />
66 000<br />
45 000<br />
30 000<br />
20 100<br />
14 400<br />
1 2 3 4 5 6 7 8<br />
Lane 1. Low <strong>Molecular</strong> Weight Calibration Kit<br />
Lane 2. Cell culture supernatant, starting material,<br />
diluted 20-fold<br />
Lane 3. IgM, human<br />
Lane 4. IgG<br />
Lane 5. Flow-through pool, diluted 20-fold<br />
Lane 6. Eluted IgM, fraction 8, diluted 8-fold<br />
Lane 7. Eluted IgM, fraction 9, diluted 8-fold<br />
Lane 8. Washing out unbound material,<br />
pool diluted 3-fold<br />
Fig. 19b. SDS-PAGE on PhastSystem, using PhastGel 4–15 with silver staining.<br />
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