Bio-rad Profinity eXact™ Purification and Tag Cleavage Con Manual de usuario descargar pdf (Pagina 12)

Section 7

Preparation of E. coli Lysate

For E. coli cultures expressing medium to high levels of

Profinity eXact-tagged proteins, (

≥10% of total protein),

50 ml of culture will yield sufficient material for a 1 ml

cartridge purification, and 250 ml of culture will yield

sufficient material for a 5 ml cartridge purification run.

For cultures expressing protein at low levels (£10% of

total protein), the culture volumes will need to be

determined empirically for each protein.

Lysate Preparation

1. Harvest cells by centrifugation.

2. Determine weight of cell pellet and resuspend in

10 volumes of bind/wash buffer (50 ml of culture

typically yields 0.5 g of paste, resulting in

approximately 5 ml of lysate).

3. Sonicate the lysate (on ice) for 3 minutes.

4. Centrifuge the lysate at

≥16,000 x g for 30 min at

4°C.

Fig. 5. Partioning profile. Precision Plus Protein™ molecular

weight markers were loaded in lane 1, followed by the total, soluble,

and insoluble fractions in lanes 2–4 respectively. The gel depicts

Profinity eXact-tagged Maltose Binding Protein, which partitions

into the soluble fraction and can be purified using the native

purification protocol. A representative chromatogram and gel for

the purification of this target protein is shown in Fig. 6.

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Bio-rad Profinity eXact™ Purification and Tag Cleavage Con Manual de usuario Pagina 12