For the pull-down assay 24 (link), the target protein (MsmtopoI) with a fusion tag (N-terminal 6xHistidine) was mixed with M. smegmatis mc2 155 soluble cell extract for 2 hours at 4°C, and then immobilized on a HisPur Cobalt agarose resin (Thermofisher). The resin with bound target proteins was washed three times in a pull-down wash buffer (10 mM HEPES, pH 7.5, 10 mM imidazole, 0.005% Tween-20) to remove the weakly retained proteins. The proteins that remained bound to the resin were eluted with pull-down elution buffer (10 mM HEPES, pH 7.5, 350 mM imidazole), separated by SDS-PAGE, and stained with coomassie blue. The protein bands of interest were excised for identification by tryptic digest and nano LC/MS/MS.
Hispur cobalt agarose resin
Manufactured by Thermo Fisher Scientific
HisPur Cobalt agarose resin is a prepacked chromatography medium designed for the purification of histidine-tagged recombinant proteins. The resin consists of cobalt-charged iminodiacetic acid (IDA) ligands covalently coupled to a highly cross-linked agarose matrix.
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Lab products found in correlation
2 protocols using hispur cobalt agarose resin
1
Identifying Protein Interactors via Pull-Down Assay
2
Protein Interaction Analysis of TopA and CobB
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Purified recombinant TopA protein (5 nM) was mixed with purified N-terminal His-tagged CobB protein (10–50 nM) in pull-down buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 0.005% v/v Tween-20) overnight at 4°C. HisPur Cobalt Agarose resin (Thermofisher), pre-equilibrated in pull-down buffer, was then added to the CobB-TopA reaction and mixed at 4°C for 2 h. After centrifugation of the reactions, the resin was washed three times in pull-down buffer with 10 mM imidazole. The proteins bound to the resin were eluted in SDS sample buffer by boiling for 2 min. Eluates were electrophoresed in 10% polyacrylamide SDS gel, and TopA was detected by western blotting with monoclonal antibodies against E. coli TopA.
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