Journal: Analytical biochemistry
Article Title: In-gel refolding allows fluorescence detection of fully denatured GFPs after SDS-PAGE.
doi: 10.1016/j.ab.2025.115861
Figure Lengend Snippet: Fig. 5. Application of the refolding procedure to squalene monooxygenase (SM) fused with three different GFP variants. The SDS-PAGE samples were prepared from HEK293 cells stably expressing SM-FLAG-tGFP or HEK293 cells transiently expressing either SM-FLAG-EGFP or SM-FLAG-msfGFP (sfGFP-V206K). A. In-gel refolding and subsequent Western blotting of SM-GFPs. The SDS-PAGE samples were heated at the indicated temperature for 5 min and resolved on SDS-PAGE (10 % gel). The gel was imaged before and after refolding in the optimized refolding buffer (1 % αCD and 20 % MeOH in Tris-Gly buffer, 30 min at room temperature). The gel after refolding was equilibrated in Towbin transfer buffer supplemented with 0.05 % SDS for 15 min, and proteins were wet-transferred to the PVDF membrane with the same transfer buffer. The membrane was probed with an anti-FLAG antibody and then re-probed with an anti-SM antibody after stripping. The filled and open arrowheads represent the denatured 95 kDa band and the non-denatured 77 kDa band, respectively. B. Quantification of the non-denatured and the denatured bands. The gray solid lines represent the fluorescent intensity of the lower, native band, and the magenta dashed lines represent the fluorescent intensity of the upper, denatured band. In-gel fluorescence was normalized to the sum of the two bands observed for the non-boiled (RT) samples. Data are mean ± SEM (n = 5 independent refolding experiments).
Article Snippet: Proteins were transferred from the gel after in-gel renaturation, which was equilibrated with a standard Towbin transfer buffer (25 mM Tris, 192 mM Gly, 20 % (v/v) MeOH) supplemented with 0.05 % SDS for 10 min, to a PVDF membrane equilibrated with the standard Towbin transfer buffer in a Mini-protean tetra wet transfer system (Bio-Rad) with myPowerII 500 power supply (AE-8155, ATTO).
Techniques: SDS Page, Stable Transfection, Expressing, Western Blot, Membrane, Stripping Membranes, Fluorescence
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