ALOD4 construct was generously provided by the lab of Dr Arun Radhakrishnan. Recombinant His-tagged ALOD4 and OlyA were purified as previously described (16 (link)). Briefly, ALOD4 expression was induced with 1 mM IPTG in OD0.5 BL21 (DE3) pLysS E. coli for 16 h at 18°C. Cells were lysed and His-ALOD4 and His-OlyA were isolated by nickel purification followed by size exclusion chromatography (HisTrap-HP Ni column, Tricorn 10/300 Superdex 200 gel filtration column; FPLC AKTA, GE Healthcare). Protein-rich fractions were pooled and concentration was measured using a NanoDrop instrument.
Histrap hp ni column
Manufactured by GE Healthcare
The HisTrap-HP Ni column is a pre-packed affinity chromatography column designed for the purification of histidine-tagged recombinant proteins. The column utilizes a Ni Sepharose High Performance matrix to capture the target protein, which can then be eluted with an imidazole-containing buffer.
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Lab products found in correlation
2 protocols using histrap hp ni column
1
Recombinant ALOD4 and OlyA Purification
2
Cloning and Expression of TrSOX Gene
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The TrSOX gene (GeneBank: ACM06094.1) was cloned in a pMA5-Pxyl-trsox plasmid and kept in our laboratory. The HisTrap HP Ni-column was purchased from GE Healthcare; AmiconUltra centrifugal filter tube (10 kDa) was from Merck Millipore; Thin-layer chromatography (TLC) plates (Merck, 105626, 105729, 105721 and 105554) were purchased from Merck Millipore; Q5 Site-Directed Mutagenesis Kit was from NEB Inc.; NeXtal Tubes JCSG Core Suites I-IV, NeXtal Tubes Classics Suite, NeXtal Tubes Classics II Suite, and NeXtal Tubes Cryos Suite were from Qiagen; and Pierce BCA Protein Assay Kit was from Thermo Scientific. N-methyl or alkaloid substrates were from MACKLIN and ALADDIN. Other reagents and chemicals were purchased from local companies and were of superior analytical grade.
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