Affinity purification of Protein A-tagged bait proteins was carried out as described in (23 (link)). Frozen cell grindate was rapidly thawed into TBT150 buffer with 1 mM Dithiothreitol (DTT). The resulting lysate was vortexed for 1 min, polytroned for 30 s and then cleared by centrifugation at 2600 g for 5 min at 4°C. Magnetic beads (Dynabeads M-270, Invitrogen), conjugated with rabbit IgG Ab (Sigma), were washed three times with TBT150 + DTT and added to the cleared cell lysate at a concentration of 3.75 mg (25 μl slurry)/0.5 g of cell grindate. The samples were rotated for 30 min at 4°C. After binding, the beads were magnetically harvested and then quickly washed three times with 10ml of TBT150 + DTT, and finally once with 10ml of LWB while vortexing at very low speed for 5 min.
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Affinity Purification of Protein A-Tagged Bait Proteins
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Ab igg
Affinity purification
Buffer
Cell
Centrifugation
Dithiothreitol
Frozen
G cell
Protein proteins
Rabbit
Corresponding Organization :
Other organizations : Université de Montréal, McGill University
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- Affinity purification of Protein A-tagged bait proteins
- Conjugation of magnetic beads with rabbit IgG Ab
- Binding of cleared cell lysate to magnetic beads
- Frozen cell grindate was rapidly thawed into TBT150 buffer with 1 mM Dithiothreitol (DTT)
- Vortexing for 1 min, polytron for 30 s, and centrifugation at 2600 g for 5 min at 4°C to clear the cell lysate
- Washing of magnetic beads three times with TBT150 + DTT
- Rotation of samples for 30 min at 4°C
- Washing of beads three times with TBT150 + DTT, and once with 10ml of LWB while vortexing at very low speed for 5 min
- Not explicitly mentioned
- Not explicitly mentioned
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