superdex s200  (Amersham Life Sciences Inc)

Journal: iScience

Article Title: Small-molecule activators of the Staphylococcus aureus ClpC/ClpP AAA+ protease

doi: 10.1016/j.isci.2026.117738

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Article Snippet: Superdex S200 , Cytiva , 28–9893–35.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Cloning, Mutagenesis, Sequencing, Software, Analysis, Microscopy

Journal: Nature Communications

Article Title: Role of PER-WDR5 interactions in the mammalian circadian clock

doi: 10.1038/s41467-026-77583-0

Figure Lengend Snippet: a Domains (yellow) and interaction partners (blue) of PER1, PER2 and WDR5. Red boxes indicate potential WDR5 binding WIN (ARART) - and WBM (L/IDVT) motifs in PER1/2. PER1/2 have two PAS (PER-ARNT-SIM) domains (PAS-A, PAS-B) to dimerize and interact with other proteins. The middle and C-terminal regions of PER1/2 interact with CK1δ/ε and CRY1/2, respectively. WDR5 consists of 7 WD repeats (WD1-7) and has two binding sites to interact with proteins containing a WIN- or WBM motif. b Alignment of WDR5 binding motifs. WIN motifs have a conserved central arginine (bold), WBM motifs consist of a conserved aspartate (bold) flanked by aliphatic amino acids. c Analytical SEC (S75 10/300) of WDR5 22-334 and PER2 1132-1252. Top: chromatograms of WDR5 22-334 (black), PER2 1132-1252 (blue), and PER2/WDR5 complex (red). Bottom: SDS-PAGE of PER2/WDR5 complex SEC. M: molecular weight marker. The PER2/WDR5 complex elutes earlier (10 mL) than free WDR5 (>11 mL). SEC results are representative of n = 3 independent experiments. d Analytical SEC (S200 10/300) of WDR5 22-334 and PER1 1013-1291. Top: chromatograms of WDR5 22-334 (black), PER1 1013-1291 (blue) and PER1/WDR5 complex (red). Bottom: SDS-PAGE of PER1/WDR5 complex SEC. The PER1/WDR5 complex elutes earlier (~13 mL) than free WDR5 (>16 mL). SEC results are representative of n = 3 independent experiments. Source data for c , d are provided as a file.

Article Snippet: The tag-free CRY1/2-PHR protein was further purified via SEC using a Superdex S200 10/300 column (Cytiva) with a buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 5% Glycerol, and 2 mM DTT.

Techniques: Binding Assay, SDS Page, Molecular Weight, Marker

Journal: Nature Communications

Article Title: Role of PER-WDR5 interactions in the mammalian circadian clock

doi: 10.1038/s41467-026-77583-0

Figure Lengend Snippet: a – c Crystal structure of WDR5 22-334 (green) bound to the PER2 1198-1211 WBM motif peptide (cyan). a Overview of the PER2/WDR5 complex. b Close-up view of the PER2/WDR5 interaction site. c Schematic representation of PER2-WDR5 interactions. Hydrogen bonds were defined by a maximum donor-acceptor distance of 3.1 Å, hydrophobic interactions by a maximum distance of 4.1 Å. ITC experiments of wildtype (WT) PER2 1132-1252 (CBD) with d WDR5 22-334 (titrant WDR5) and g CRY1 1-496 (PHR) (titrant PER2). Data were fit with a one-site binding model (lower panel). ITC results are representative of n = 3 independent experiments. ITC results (K D values) for interactions of PER2 1132-1252 (WT, I1205G, D1206A, V1207A) with e WDR5 22-334 (WT, N225A) and h CRY1-PHR (mean K D values ± SD from triplicates) (Supplementary Table and Supplementary Figs. –g, , b–d). f Analytical SEC (S200 10/300) of PER2 1132-1252, RbBP5 and WDR5 22-334. Top: chromatograms of PER2/WDR5 (red)- and RbBP5/WDR5 (magenta) complexes (1:3 excess WDR5) and of a competition experiment of a preformed PER2/WDR5 complex (PER2:WDR5) with added equimolar RbBP5 (brown). Bottom: SDS-PAGE of the competition experiment (SEC peak fractions). No trimeric PER2/WDR5/RbBP5 complex was observed, but only the dimeric WDR5/RbBP5 complex. PER2 is displaced by RbBP5. SEC results are representative of n = 2 independent experiments. i Close up view of PER2 WBM motif region (cyan) bound to CRY1-PHR (red) (PDB: 4CT0 ,). j SEC analysis (S200 10/300) of WDR5 22-334, CRY1 1-496 and PER2 1132-1252. Top: chromatograms of WDR5 (black), CRY1 (magenta) and of a preformed PER2/WDR5 complex (PER2:WDR5) mixed with equimolar CRY1 (green). Bottom: SDS-PAGE of SEC run with the PER2:WDR5 + CRY1 mixture. No trimeric PER2/WDR5/CRY1 complex was observed, but only a PER2/CRY1 complex eluting at 14.3–15.1 mL. WDR5 is displaced by CRY1 and co-elutes with monomeric WDR5 (16.3–17.5 mL). SEC results are representative of n = 2 independent experiments. Source data for d , f , g , j are provided as a file.

Article Snippet: The tag-free CRY1/2-PHR protein was further purified via SEC using a Superdex S200 10/300 column (Cytiva) with a buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 5% Glycerol, and 2 mM DTT.

Techniques: Binding Assay, SDS Page, Analysis

Journal: Nature Communications

Article Title: Role of PER-WDR5 interactions in the mammalian circadian clock

doi: 10.1038/s41467-026-77583-0

Figure Lengend Snippet: ITC analyses of the interaction of PER1 1013-1291 with a WDR5 22-334 (titrant WDR5) and b CRY1 1-496 (PHR) (titrant PER1). Data were fit with a one-site binding model (lower panel). ITC results are representative of n = 3 independent experiments. c ITC results ( K D ) for interactions of PER1 1013-1291 (WT, R1111A, D1197A, R1111A D1197A, R1111A R1113A) with WDR5 22-334 (WT, N225A) or CRY1 1-496 (mean K D values ± SD from triplicates) (Supplementary Table , Supplementary Fig. a–d and 5a–c). ITC results are representative of n = 3 independent experiments. d SEC (S200 10/300) analyses of the interaction of PER1 1013-1291 with WDR5 22-334 WT (top), the WIN site triple mutant WDR5 D107A F133A Y260A, and the WIN/WBM site mutant WDR5 D107A F133A Y260A N225A. The SEC run of PER1 alone is shown for comparison (bottom). Left: SDS-PAGE analyses of peak SEC fractions, right: SEC chromatograms of the PER1/WDR5 complex (red), PER1 alone (blue), and WDR5 alone (black). SEC results are representative of n = 2 independent experiments. e Chemical structure of the WDR5 WIN site inhibitors Inh-4 and Inh-5 . f Inh-4 and Inh-5 weaken the PER1 1013-1291 interaction with WDR5 22-334 in SEC (S200 10/300). Left: Chromatograms of the PER1/WDR5 complex without (red) and in presence of Inh-4 (brown) or Inh-5 (green). PER1 alone (blue) and WDR5 (black arrow) are shown for comparison. Right: SDS-PAGE analyses. SEC results are representative of n = 2 independent experiments. Source data for a , b , d , f are provided as a file.

Article Snippet: The tag-free CRY1/2-PHR protein was further purified via SEC using a Superdex S200 10/300 column (Cytiva) with a buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 5% Glycerol, and 2 mM DTT.

Techniques: Binding Assay, Mutagenesis, Comparison, SDS Page

Journal: Nature Communications

Article Title: Role of PER-WDR5 interactions in the mammalian circadian clock

doi: 10.1038/s41467-026-77583-0

Figure Lengend Snippet: a Inh-5 disrupts and Inh-4 weakens the interaction of hMLL1 3748-3969 (WIN-SET) with WDR5 22-334 in SEC (S200 10/300). Chromatograms (top) and SDS-PAGE analyses (bottom) of the MLL1/WDR5 complex without (orange) and in presence of Inh-4 (brown) or Inh-5 (green). Free MLL1 (violet) and WDR5 (black arrow) are shown for comparison. SEC results are representative of n = 2 independent experiments. b Analytical SEC (S200 10/300) of WDR5 22-334, PER1 1013-1291 and hMLL1 3748-3969. Top: chromatograms of PER1/WDR5 (red)- and MLL1/WDR5 (orange) complexes and of the competition experiment of a preformed MLL1/WDR5 complex (MLL1:WDR5) with added equimolar PER1 (light blue). Bottom: SDS-PAGE of the competition experiment. Newly formed PER1/WDR5 complex elutes at ~ 12.5 mL, remaining MLL1/WDR5 complex at ~ 15 mL. PER1 partially displaces MLL1 (~16.5 mL) from the preformed MLL1/WDR5 complex. SEC results are representative of n = 3 independent experiments. c Analytical SEC (S200 10/300) of equimolar PER1 1013-1291, RbBP5 and WDR5 22-334. Top: chromatograms of PER1/WDR5 (red)-, RbBP5/WDR5 (pink) and PER1/WDR5/RbBP5 (brown) complexes. Bottom: SDS-PAGE of the trimeric PER1/WDR5/RbBP5 complex (SEC peak fractions), that eluted significantly earlier than the dimeric complexes and the individual PER1 and WDR5 proteins (Fig. ). Note that a different SEC S200 10/300 column was used in Fig. and c than in all other experiments stating S200 10/300. SEC results are representative of n = 2 independent experiments. d Analytical SEC (S200 10/300) of PER1 1013-1291, PER2 1132-1252 and WDR5 22-334. Chromatogram (top) and SDS-PAGE (bottom) of the SEC run with the PER1 + WDR5 + PER2 mixture. No trimeric PER1/WDR5/PER2 complex was observed, but only dimeric PER1/WDR5- and PER2/WDR5 complexes. Arrows indicate elution volumes of individual PER1/WDR5- and PER2/WDR5 complexes and of WDR5. SEC results are representative of n = 2 independent experiments. e Chromatograms and SDS-PAGE analyses of SEC (S200 10/300) of the PER1 1013-1291 interaction with CRY1 1-496 (PHR) and WDR5 22-334. SDS-PAGE top: equimolar mixture of PER1, CRY1, and WDR5, 1 h incubation (dark green chromatogram); second: preformed PER1/WDR5 complex (ON), subsequently incubated 1 h with equimolar CRY1 (light green chromatogram). Bottom: dimeric PER1/CRY1 and PER1/WDR5 complexes (orange and red chromatograms) start to elute at 11.2 and 11.4 mL. Some trimeric PER1/WDR5/CRY1 complex is formed, that starts to elute before 11.2 ml. Non-incorporated WDR5 elutes after 16 ml. SEC results are representative of n = 3 independent experiments. f Schematic of PER-WDR5 interactions and their interplay with CRY, RbBP5 and MLL1. PER2 only binds to the WBM site of WDR5, and another protein could bind to the WDR5 WIN site. PER1 binds to both WDR5 binding sites via its WIN- and WBM motifs, but the WIN site interaction is more important. Alternatively, PER1 can only bind to the WIN site, while another protein binds to the WBM site, for example, RbBP5. Thereby, PER1 could replace MLL1 on the WIN site. PER2 binds to either WDR5 or CRY, whereas PER1 can form a trimeric PER1/WDR5/CRY complex. PER1/WDR5/RbBP5- and PER1/WDR5/CRY complexes could be formed at the transition from the active state (MLL1 bound) to the early repressed state (CRY/PER/WDR5 containing repressive complexes) of CLOCK/BMAL1. PER/WDR5 complex formation requires excess PER or spatial/temporal separation from CRY. Source data for a – e are provided as a file.

Article Snippet: The tag-free CRY1/2-PHR protein was further purified via SEC using a Superdex S200 10/300 column (Cytiva) with a buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 5% Glycerol, and 2 mM DTT.

Techniques: SDS Page, Comparison, Incubation, Binding Assay

Journal: STAR Protocols

Article Title: Protocol for the purification, analysis, and handling of acyl carrier proteins from type I fatty acid and polyketide synthases

doi: 10.1016/j.xpro.2025.103762

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Article Snippet: Cytiva S200 10/300 size exclusion column , Cytiva , Cat# 28990944.

Techniques: Virus, Recombinant, Plasmid Preparation, Software