superose 6 increase 10 30 size exclusion chromatography column  (GE Healthcare)

Journal: Journal of Cell Science

Article Title: Microtubule binding of the human augmin complex is directly controlled by importins and Ran-GTP

doi: 10.1242/jcs.261096

Figure Lengend Snippet: Purification and characterization of the human augmin complex. (A) Schematic of the eight subunits of the augmin complex. HAUS1 carries a C-terminal 3C-His 6 -tag, and HAUS2 has a C-terminal mGFP-TEV-TwinStrep tag. Numbers indicate amino acid positions. (B) Absorbance profile of the human augmin complex eluting from a Superose 6 Increase 10/30 size-exclusion chromatography column. Absorbances at 260, 280 and 455 nm are indicated in purple, blue and orange, respectively. The complete complex used for experiments in this study elutes in fractions E2–E3 (orange box). Fractions E5–E7 (brown box) contain mostly subcomplexes. mAU, the milli-absorbance unit. (C) SYPRO Ruby-stained SDS-PAGE gel of the fractions eluted from the size-exclusion chromatography column. Orange and brown boxes indicate separately pooled fractions. HAUS2* indicates HAUS2 not having undergone TEV cleavage of the affinity tag. Positions of molecular mass markers are show in kDa. (D) Negative-stain electron microscopy of the different pools: fractions E2–E3 contain the complete complex, and fractions E5–E7 also contain subcomplexes. Data in B–C are representative of three experiments. Data in D are representative of five (E2-E3) or two (E5-E7) experiments.

Article Snippet: The solution was then passed over a Superose 6 Increase 10/30 size-exclusion chromatography column (GE Healthcare Life Sciences) that was pre-equilibrated in 50 mM phosphate buffer pH 7.5, 250 mM NaCl, 4 mM MgCl 2 , 3% glycerol, 0.5 mM TCEP.

Techniques: Purification, Size-exclusion Chromatography, Staining, SDS Page, Electron Microscopy