Journal: Protein expression and purification

Article Title: Optimized mammalian expression system for the ubiquitin E3 ligase E6AP/UBE3A

doi: 10.1016/j.pep.2025.106661

Figure Lengend Snippet: (A ) Immunoblot analysis with anti-His, anti-E6AP, or anti-β-actin (as a loading control) antibodies of whole cell lysates from untransfected Expi293 cells (lane 1) or 24 (lane 2), 48 (lane 3), or 72 (lane 4) hours post-transfection with UBE3A-thrombin-StrepII-His plasmid. Cells were grown in ExpiMedia. 10 μg of total protein was added to each lane. (B) Immunoblot analysis with anti-His, anti-E6AP, or anti-β-actin (as a loading control) antibodies of whole cell lysate from Expi293 cells transfected with varying quantities of UBE3A-thrombin-StrepII-His plasmid as indicated without (−) or with (+) the addition of enhancer. 10 μg of total protein was added to each lane. A schematic for the experimental timeline is included at the top.

Article Snippet: In addition to its role in carcinogenesis, E6AP plays a critical role in neurobiological function and its dysfunction is associated with Angelman Syndrome (AS) and autism spectrum disorders [ 17 – 19 ].

Techniques: Transfection, Expressing, Western Blot, Control, Plasmid Preparation

Journal: Protein expression and purification

Article Title: Optimized mammalian expression system for the ubiquitin E3 ligase E6AP/UBE3A

doi: 10.1016/j.pep.2025.106661

Figure Lengend Snippet: (A ) Immunoblot analysis with anti-His, anti-E6AP, or anti-β-actin (as a loading control) antibodies of whole cell lysates from growth in FreeStyle media for untransfected Expi293 cells (lane 1) or 4 (lane 2), 8 (lane 3), 12 (lane 4), 24 (lane 5), or 48 (lane 6) hours post-transfection with UBE3A-thrombin-StrepII-His plasmid. 10 μg of total protein was added to each lane. (B) Immunoblot analysis with anti-His, anti-E6AP, or anti-β-actin (as a loading control) antibodies of whole cell lysates from growth in 300 mL FreeStyle media for untransfected Expi293 cells and 24 hours post-transfection with UBE3A-thrombin-StrepII-His plasmid (lane 2). 10 μg of total protein was added to each lane. (C ) Fractions subjected to SDS-PAGE during affinity chromatography purification by batch mode of E6AP from Expi293 cells. Lane 5 shows E6AP bound to streptavidin resin by a StrepII tag at its C-terminal end. E6AP lost in wash steps is shown in lanes 7 – 11. Samples from stepwise elutions with 50 mM biotin are shown in lanes 13 through 16. ( D) E6AP-containing fractions resolved by SDS-PAGE following size exclusion chromatography purification. The samples of lanes 13 – 16 of panel A were mixed and injected into an FPLC system for further purification. The final sample was generated by combining fractions 6 through 10.

Article Snippet: In addition to its role in carcinogenesis, E6AP plays a critical role in neurobiological function and its dysfunction is associated with Angelman Syndrome (AS) and autism spectrum disorders [ 17 – 19 ].

Techniques: Purification, Size-exclusion Chromatography, Western Blot, Control, Transfection, Plasmid Preparation, SDS Page, Affinity Chromatography, Injection, Generated

Journal: Protein expression and purification

Article Title: Optimized mammalian expression system for the ubiquitin E3 ligase E6AP/UBE3A

doi: 10.1016/j.pep.2025.106661

Figure Lengend Snippet: (A) Mass photometry profile of the E6AP sample plotting detected counts against mass. E6AP was diluted 6-fold with Tris-buffer and thoroughly mixed yielding a well content concentration of 200 nM. A predominate signal at 88 kDa (70%) was observed, along with broadened peaks at 178, 270, and 393 kDa. Lower molecular weight impurities appear at 3% population. (B, C) Q-TOF mass spectrometry experiment for E6AP showing a UV trace at 280 nm ( B ) and deconvoluted mass ( C ). 10 μL of pure E6AP was injected into a Q-TOF mass spectrometer and absorbance recorded at 280 nm to find a single peak at 6 minutes following injection. The major peak at 6 minutes was analyzed to yield a predominate signal with a deconvoluted mass of 101.2 kDa, consistent with the expected molecular weight for E6AP in its monomeric state. ( D ) Immunoblot for ubiquitin or E6AP of 1 μg or 4 μg of E6AP sample. ( E ) Immunoblots for ubiquitin (top) or E6AP (bottom) for combinatorial mixtures of E6AP, E1, E2, ubiquitin, ATP, and vOTU, as indicated.

Article Snippet: In addition to its role in carcinogenesis, E6AP plays a critical role in neurobiological function and its dysfunction is associated with Angelman Syndrome (AS) and autism spectrum disorders [ 17 – 19 ].

Techniques: Activity Assay, Concentration Assay, Molecular Weight, Mass Spectrometry, Injection, Western Blot, Ubiquitin Proteomics