gradient denaturing sds page gel  (Bio-Rad)

Journal: EMBO Reports

Article Title: TRIM2 E3 ligase substrate discovery reveals zinc-mediated regulation of TMEM106B in the endolysosomal pathway

doi: 10.1038/s44319-025-00667-3

Figure Lengend Snippet: ( A ) Domain organization and corresponding cellular location of TMEM106B depicted diagrammatically. ( B ) In vitro ubiquitination assays involving the enzymatic cascade components and the N-terminal cytosolic region of TMEM106B (1-95). Minor impurities from protein degradation during purification are marked with an asterisk. The samples produced were assessed through Coomassie stained SDS-PAGE (left), immunoblots against ubiquitin (middle), and TMEM106B (right). Note that the antibody against ubiquitin cross-reacts with the E2 (compare lanes 6 and 7). ( C ) Similar ubiquitination assays with varying TMEM106B 1-95 concentration, from 80 to 5 µM (final). The presence of two high MW bands upon addition of ATP shows that both mono- and di-ubiquitination can occur. ( D ) The region corresponding to ubiquitinated TMEM106B was excised from the polyacrylamide gel and analyzed for PTMs with MS. Shown are the modification sites identified along the TMEM106B protein sequence, with the amino acids involved indicated below using one-letter codes. Note that save for the Gly-Gly modification, all other PTMs occur due to sample preparation prior to MS. These results could be replicated at least three times in the laboratory. .

Article Snippet: The cleared lysate from cellular experiments or samples from in vitro assays (~2–50 μg) were run on a gradient denaturing SDS-PAGE gel (Mini-Protean TGX, BioRad) and transferred to a polyvinylidene difluoride membrane using the Trans-Blot Turbo transfer system according to manufacturer’s instructions (BioRad).

Techniques: In Vitro, Ubiquitin Proteomics, Purification, Produced, Staining, SDS Page, Western Blot, Concentration Assay, Modification, Sequencing, Sample Prep

Journal: EMBO Reports

Article Title: TRIM2 E3 ligase substrate discovery reveals zinc-mediated regulation of TMEM106B in the endolysosomal pathway

doi: 10.1038/s44319-025-00667-3

Figure Lengend Snippet: ( A ) Overlaid 1 H- 15 N HSQC spectra of 15 N-labeled TMEM106B C61S/C64S in the absence and presence of 5-fold molar TRIM2 RBCC . Shown as insets are zoomed-in views of three sample amide peaks. ( B ) Line broadening upon addition of 5-fold molar excess TRIM2 RBCC was quantified as the ratio of peak heights in the presence and absence of TRIM2 RBCC (I RBCC /I 0 ), for TMEM106B WT and TMEM106B C61S/C64S (see also Fig. ). The ratios are plotted as a function of the magnitude rank from low to high values. The error bars correspond to the cumulative error arising from the two spectra used for quantification, obtained from signal-to-noise ratio estimates using NMRFAM-Sparky ( n = 1). ( C ) Ubiquitination of TMEM106B was monitored using SDS-PAGE and immunoblotting as described above, comparing dimeric and monomeric variants. Note that although equal amounts of substrate were used, the antibody recognizes TMEM106B C61S/C64S more poorly, giving rise to weaker signals (compare SDS-PAGE and Western blots). Biophysical measurements were performed once, while biochemical experiments could be replicated at least three independent times in the laboratory. .

Article Snippet: The cleared lysate from cellular experiments or samples from in vitro assays (~2–50 μg) were run on a gradient denaturing SDS-PAGE gel (Mini-Protean TGX, BioRad) and transferred to a polyvinylidene difluoride membrane using the Trans-Blot Turbo transfer system according to manufacturer’s instructions (BioRad).

Techniques: Labeling, Ubiquitin Proteomics, SDS Page, Western Blot