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Tandem affinity purification of M. tuberculosis proteins.

SB Samuel H. Becker
KU Kathrin Ulrich
AD Avantika Dhabaria
BU Beatrix Ueberheide
WB William Beavers
ES Eric P. Skaar
LI Lakshminarayan M. Iyer
LA L. Aravind
UJ Ursula Jakob
KD K. Heran Darwin
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Purifications of TAP-tagged proteins from M. tuberculosis were performed under low-salt conditions as described (66). The following changes were made to the protocol: 100 μl of packed Ni-NTA beads and 100 μl of M2 anti-FLAG affinity gel were used; 100 μl of 100 μM 3 × FLAG peptide was used for the final elution. For capturing RucTAP interactions with other M. tuberculosis proteins, M. tuberculosis lysates were incubated at 45°C for 10 min either in the absence (Fig. 5C, lane 1) or presence (Fig. 5C, lane 2) of 2 mM H2O2 and 50 μM CuCl2; purifications were subsequently performed as described above. Samples were boiled in 4× reducing SDS sample buffer prior to running SDS-PAGE gels.

Copyright and License information:  ©2020 Becker et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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