pLD‐CoHSA‐Trxm

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The resulting plasmids, termed pET Trxm and pET Trxf, were introduced into E. coli (BLR DE3, Novagene)	Overexpressed proteins were purified by chromatography in affinity columns packed with Ni‐NTA agarose (Qiagen, Hilden, Germany). Specific anti‐Trx m and anti‐Trx f antibodies were raised by immunizing rabbits with purified His‐tagged proteins (Abyntek, Bilbao, Spain). Antibody cross‐reactivity was eliminated by affinity purification (exclusion) of anti‐Trx m IgG with the Trx f antigen and vice versa.

Protocol tips
The resulting plasmids, termed pET Trxm and pET Trxf, were introduced into E. coli (BLR DE3, Novagene)
Downstream tips
Overexpressed proteins were purified by chromatography in affinity columns packed with Ni‐NTA agarose (Qiagen, Hilden, Germany). Specific anti‐Trx m and anti‐Trx f antibodies were raised by immunizing rabbits with purified His‐tagged proteins (Abyntek, Bilbao, Spain). Antibody cross‐reactivity was eliminated by affinity purification (exclusion) of anti‐Trx m IgG with the Trx f antigen and vice versa.

Publication protocol

Tobacco Trx m and Trx f were expressed in E. coli as His‐tagged polypeptides. The coding sequences were amplified by PCR with the same primers as those used for their cloning (FusTrxm/f‐For and CoTrxm/f‐Rev); the resulting PCR fragment was digested with NcoI and NotI and cloned in a pET 28a(+) vector (Novagene; Merck KGaA, Darmstadt, Germany). The resulting plasmids, termed pET Trxm and pET Trxf, were introduced into E. coli (BLR DE3, Novagene). Overexpressed proteins were purified by chromatography in affinity columns packed with Ni‐NTA agarose (Qiagen, Hilden, Germany). Specific anti‐Trx m and anti‐Trx f antibodies were raised by immunizing rabbits with purified His‐tagged proteins (Abyntek, Bilbao, Spain). Antibody cross‐reactivity was eliminated by affinity purification (exclusion) of anti‐Trx m IgG with the Trx f antigen and vice versa.

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