GE healthcare
Calmodulin Sepharose 4B, 10 mL
17052901
The aim of our experiment is to purify TAP-tagged SPT16 from S. cerevisiae cells expressing TAP-SPT16. TAP tag contains protein and calmodulin binding protein separated by TEV protease cleavage site. Yeast cells were lysed by bead beater and the lysate was bound to IgG beads. After cleavage with TEV protease, the protein eluted was bound to 500 µL of calmodulin sepharose 4B. TAP-SPT16 bound to calmodulin sepharose 4B was eluted by using elution buffer. Elutions were examined for TAP-SPT16 by running on SDS-PAGE and staining with Coomassie Brilliant Blue R-250.
Protein purification
S. cerevisiae whole cell lysate
Yeast cells were harvested by centrifugation. Resuspended cells in extraction buffer and lysed cells by bead beating. Spun down cell debris by centrifugation. The supernatant was ultracentrifuged for 1.5 hr at 45000 rpm at 4°C. Clarified extract was incubated with IgG resin overnight at 4°C. Beads were incubated with TEV protease o/n and eluted TEV cleaved products. TEV cleavage was incubated with calmodulin resin o/n on the mixer. Protein bound to calmodulin resin was eluted with a calmodulin elution buffer at room temperature. Analyzed eluted fraction by SDS-PAGE and Coomassie staining.
All the steps for purification has to be carried out at 4°C. More cells are required if your require more protein for analysis.
TAP-SPT16 was purified without any non-specific proteins. SPT16 is a part of FACT complex which also contains Pob3 protein. We could see the presence of Pob3 in eluates by Coomassie staining.
N/A
Even though our product was old, still the purification was perfect.
Excellent product for purifying TAP-tagged proteins.