Protein Protocols Protein Protocols

1126 Schwarz
3.5. General Purification of IgG Using MEP-HyperCell (see Note 5)
This protocol is designed for the purification of all IgG from hybridoma cell culture
supernatant and ascitic fluids. For cell culture supernatants that contain more than 5%
fetal calf serum, the purity is relatively low at approx 50–65%.
1. Bring all materials to room temperature.
2. Equilibrate a 10-mL MEP-HyperCell gel with 5 column volumes of buffer A.
3. Load supernatant onto the column at 5 mL/min.
4. Load up to 20 mg of antibody/mL of gel.
5. Wash with 10 column volumes of buffer A.
6. Elute antibody with 8 column volumes of buffer B.
7. Reequilibrate the column with 2 column volumes of buffer A.
8. Wash with 8 column volumes of buffer E followed by 10 column volumes of buffer A.
The column is ready for the next chromatography run.
3.6. Immobilized Metal Affinity Purification of IgG (see Note 6)
This protocol is designed for the purification of all IgG from hybridoma cell culture
supernatant and ascitic fluids. For cell culture supernatants that contain more than 5%
fetal calf serum, the monoclonal antibody will be contaminated with bovine IgG.
1. Bring all materials to room temperature.
2. Dilute three parts of supernatant with one part of buffer C.
3. Equilibrate a 10-mL IMAC gel with 5 column volumes of buffer B.
4. Load the column with buffer E until the column is completely colored.
5. Wash with 5 column volumes of buffer B.
6. Equilibrate with 5 column volumes of buffer A.
7. Load supernatant onto the IMAC gel column at 5 mL/min.
8. Load up to 10 mg of antibody/mL of gel.
9. Wash with buffer A for 15 column volumes until the UV baseline is reached.
10. Elute antibody with 10 column volumes of buffer B.
11. Strip column with 5 column volumes of buffer D, followed by 5 column volumes of buffer B.
The column is ready for the next chromatography run.
3.7. Concluding Remarks
All the protocols provided in the yield antibodies in good yield and good purity. If
not further specified in the accompanying notes, the protocols are usable with virtually
all commercially available gels. Difficulties in using a protein A gel can be overcome
by using a different affinity column such as a thiophilic gel or an IMAC gel. In most
protocols provided, elution is accomplished at acidic pH values. Therefore, further
purification can be accomplished by adsorbing the eluate onto a cation-exchange gel.
Most of the impurities will flow through and, when using gradient elution, the antibody
is generally the first protein to elute from the column.
4. Notes
1. This general protocol works well with high-affinity antibodies and is independent of the
particular protein A gel used. The purity of the antibody recovered can be increased by
first dialyzing the supernatant or the ascitic fluid against buffer A. Sometimes, phenol red