Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2014 May 7;9(5):e96809. doi: 10.1371/journal.pone.0096809

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2014 Kozłowska et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

PMC Copyright notice

(A) Schematic illustration of recombinant DmChd64 and TcChd64. Recombinant constructs for DmChd64 and TcChd64 were prepared. DmChd64/TcChd64was N-terminally tagged with the Strep II-tag, and C-terminally tagged with the 8×His-tag. DmChd64/TcChd64 was N-terminally tagged with the Strep II-tag and DmChd64/TcChd64 was C-terminally tagged with the 8×His-tag. (B) Coomasie Brilliant Blue R-250-stained SDS-PAGE analysis of the expression and purification procedure of DmChd64. Lane 1, molecular mass standards; lane 2 and 3, whole cell extracts from bacterial cells containing the pQE80L-SXH plasmid with the cDNA insert (pQE80L-SXH/DmChd64) harvested before (T_NI) and 3 h after expression induction with IPTG (T_{3 h}); lane 4, soluble protein fractions obtained after cell lysis (Sup); lane 5, the flow-through (FT) fraction obtained from the Co²⁺-TALON column; lane 6, protein fractions containing impurities washed (W) from the Co²⁺-TALON column using buffer A; lane 7, protein fractions containing impurities eluted during the imidazole gradient sub-step using buffer A and B solutions containing 20 mM imidazole (Im₂₀); lane 8, pooled fractions eluted from Co²⁺-TALON resin with buffer B containing 200 mM imidazole (Im₂₀₀); lane 9, protein fractions containing impurities washed (W) from the Strep-Trap HP column using buffer C; lane 10, pooled fractions eluted from the Strep-TrapHP column with buffer D containing 2.5 mM desthiobiotin (D_2.5); lane 11, pooled fractions of DmChd64 from the Superdex 200 10/300 GL column (S). (C) Coomasie Brilliant Blue R-250-stained SDS-PAGE analysis of the expression and purification procedure of TcChd64. Lane 1, molecular mass standards; lane 2 and 3, whole cell extracts from bacterial cells containing the pQE80L-XH plasmid with the cDNA insert (pQE80L-XH/TcChd64) harvested before (T_NI) and 3 h after expression induction with IPTG (T_{3 h}); lane 4, soluble protein fractions obtained after cell lysis (Sup); lane 5, the flow-through (FT) fraction obtained from the Co²⁺-TALON column; lane 6, protein fractions containing impurities washed (W) from the Co²⁺-TALON column using buffer E; lane 7, protein fractions containing impurities eluted during the imidazole gradient sub-step using buffer E and F solutions containing 20 mM imidazole (Im₂₀); lane 8, pooled fractions eluted from the Co²⁺-TALON resin with buffer F containing 200 mM imidazole (Im₂₀₀); lane 9, protein fractions containing impurities from the first peak from tandem connected Superdex 75 10/300 GL columns (S₁)_; lane 10, pooled fractions of TcChd64 from the two tandem Superdex 75 10/300 GL columns (S₂).