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![[Figure 4]](ai5014fig4mag.jpg)
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Figure 4
Comparison of ATAD2B expression and purification from E. coli and Sf9 cells. Left panels. Top: schematic of E. coli expression and Coomassie-stained 10% SDS–PAGE gel of peak samples from size-exclusion chromatography (SEC). The molecular-weight (MW) ladder is shown in the first lane with the mass of each band labeled. Lane 2 shows protein sample from peak 1 of the SEC chromatogram below eluting at 9 ml that contains ATAD2B along with GroEL contaminant. Lane 3 shows protein sample from peak 2 eluting at ∼15 ml that contains the GST-tag. Middle: SEC profile (Superdex 200 Increase 10/300 GL) showing a peak for ATAD2B and GroEL (9 ml). The additional peak at ∼15 ml corresponds to the cleaved GST-tag (observed as 50 kDa on SEC and 25 kDa on SDS–PAGE). Bottom: cryo-EM micrograph confirming the presence of heptameric GroEL contaminant in the purified sample. Right panels. Top: schematic of Sf9 expression and Coomassie-stained 10% SDS–PAGE gel showing greater than 90% pure ATAD2B collected from peak 1 of the SEC chromatogram below. The peak 2 lane contains the GST-tag. Middle: SEC profile (Superdex 200 Increase 10/300 GL) of the Sf9 purified sample with peaks for ATAD2B (∼9 ml) and the cleaved GST-tag (∼15 ml). Bottom: cryo-EM micrograph confirming hexameric ATAD2B in the purified sample. |
![[Figure 4]](ai5014fig4.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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