minidawn  (Waters Corporation)

Journal: Biochemical Journal

Article Title: Autoregulation of the MET receptor tyrosine kinase by its intracellular juxtamembrane domain

doi: 10.1042/BCJ20253378

Figure Lengend Snippet: ( A ) Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) analysis of the dephosphorylated KD and ICD. Elution volume from the size exclusion column is plotted on the x-axis. The left y-axis shows the light-scattering signal. Calculated molecular weights (MW) are indicated above the elution peaks, with their distribution shown on the right y-axis. ( B ) Schematic depiction of the dimeric MET constructs engineered via fusion of the translocated promoter region (TPR). ( C-D ) Representative traces from the coupled kinase assay for the indicated dephosphorylated MET constructs, obtained in the absence ( C ) or presence of substrate ( D ). Bar graphs on the right show the calculated catalytic rates from triplicate measurements for a representative experiment. Error bars represent standard deviation (SD). ( E ) ADP-Glo activity measurements for the indicated proteins are shown as fold change in relative luminescence units (RLUs), normalized to the activity of KD measured in the absence (left) or presence (right) of substrate. summarizes average values for each ADP-Glo assay from independent experiments.

Article Snippet: Dephosphorylated MET proteins (100 μl of 20 μM protein) were separated on a Shodex KW-802.5 column by High-Performance Liquid Chromatography (HPLC) with a UV detector (Shimadzu), connected with a miniDAWN MALS detector and an Optilab refractometer (Wyatt Technology) at 4°C.

Techniques: Size-exclusion Chromatography, Multi-Angle Light Scattering, Construct, Kinase Assay, Standard Deviation, Activity Assay, Glo Assay

Journal: bioRxiv

Article Title: Dimerization of human PARP15 is required for NAD + binding and automodification

doi: 10.64898/2025.12.15.694324

Figure Lengend Snippet: A) A difference in activity is seen between the WT and mutant proteins using NAD + hydrolysis assay, showing that both PARP15 CAT and PARP15 FL D665A, D665R and PARP15 CAT R576A mutants are not hydrolyzing NAD + efficiently. B) PARP15 FL D665A and D665R are modified during expression in the Sf21 insect cells, showing MARylation before the incubation with NAD + in vitro , but not showing enhanced MARylation upon NAD + incubation. Full western blot available in Supplementary (Fig. S2) . C) In SEC–MALS PARP15 FL WT elutes as a dimer, whereas D665A and D665R elute as monomers. D) PARP15 FL H559Y tagged with either GFP or HA and HA-PARP15 FL H559Y D665R were expressed in HEK293 cells. GFP-tagged proteins were enriched using GFP-TRAP. Co-purified HA-tagged proteins were evaluated on western blots. For control, whole cell lysates (WCL) were analyzed for expression of the indicated proteins. E) The expression of GFP-PARP15 FL , GFP-PARP15 FL H559Y and GFP-PARP15 FL D665R was induced with doxycycline in U2OS cells and subsequently the indicated proteins were enriched using GFP-TRAPs. PARP15 expression was monitored using specific antibodies after enrichment and in WCL. MARylation was assessed in the enriched samples.

Article Snippet: Light scattering detector Wyatt MiniDawn MALS and Wyatt Optilab refractive index (RI) detector were connected to a Shimadzu HPLC unit.

Techniques: Activity Assay, Mutagenesis, Hydrolysis Assay, Modification, Expressing, Incubation, In Vitro, Western Blot, Purification, Control