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Figure 2 | BMC Biochemistry

Figure 2

From: Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts

Figure 2

Expression of atTic20 and its truncated mutant (atTic20ΔN20) in bacterial membranes. (A) Fractionation of E. coli BL21 CodonPlus (DE3)-RIPL cells expressing recombinant atTic20 using the autoinduction method. Protein profiles of bacterial cell fractions (S, total soluble proteins; M, total membranes; I, inclusion bodies) and atTic20 purified from the membrane fraction (P) were compared using SDS-PAGE stained with Coomassie Blue. 5 μg of total protein was loaded in each lane. The left lane contains a protein molecular weight (kDa) ladder (L). (B) Western blot detection of recombinant atTic20 in the bacterial membrane fraction of auto-induced (I) and uninduced (U) bacterial cells using mouse IgG2b anti-histidine antibody, detected by chemiluminescence using a horseradish peroxidase-linked secondary antibody. (C) An assay for the membrane marker NADH oxidase was used to confirm the presence of membranes in the “membrane fraction” and to compare to the soluble protein fraction from Figure 2A. The NADH oxidase assay measures the conversion of NADH from its reduced to its oxidized form, indicated by a decrease in A340 over time. Average specific NADH oxidase activity (U/mg protein) of the soluble and bacterial membrane fractions are shown. (D) Comparison of purified atTic20 (FL) and its truncated mutant (ΔN20), resolved using semi-native PAGE (2 mM SDS in the running buffer; 0 mM SDS included in the sample buffer) stained with Coomassie blue. The molecular weights (kDa) of the protein ladder (L) are indicated.

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