Journal: Biology of the Cell
Article Title: Direct observation of fluorescent proteins in gels: A rapid, cost‐efficient, and quantitative alternative to immunoblotting
doi: 10.1111/boc.202400161
Figure Lengend Snippet: Comparison of in‐gel fluorescence (IGF) of tagRFP‐T‐, mRuby2‐, mCherry‐, and mKO‐κ‐tagged proteins. (a) Yeast expressing Bmh1‐tagRFP‐T, Bmh1‐mRuby2, Bmh1‐mCherry, or Bmh1‐mKO‐κ were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at the indicated temperatures. After migration on a commercial precast 4%–20%TGX gel (Bio‐Rad), gels were imaged for red fluorescence using a Typhoon or a Chemidoc MP, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. Proteins were then transferred to a nitrocellulose membrane and immunoblotted with anti‐Bmh1 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. * indicates the fluorescent species. (b) Yeast expressing Bmh1‐mCherry were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at the indicated temperatures in a gradient thermocycler. After migration on a commercial precast 4–20% TGX gel (Bio‐Rad), gels were imaged for red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc. Proteins were then transferred to a nitrocellulose membrane and immunoblotted with anti‐Bmh1 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. (c) Same as (b) on lysates of yeast expressing Bmh1‐mKO‐κ. (d) Quantification of the fluorescence signal as a function of the denaturation temperature for mCherry‐tagged and mKO‐κ‐tagged Bmh1 ( n = 3; ±SD). Solid line: sigmoidal fit of the data (GraphPad Prism), dotted line: 95% confidence interval of the fit. HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate.
Article Snippet: Tagging with yeast codon‐optimized Tag‐RFP‐T or mRuby2 was achieved using the pFa6‐link‐yoTag‐RFP‐T‐CaURA3 and pFA6a‐link‐yomRuby2‐SpHIS5, respectively (gifts from Kurt Thorn and Wendell Lim, Addgene plasmids #44877 and #44843).
Techniques: Comparison, Fluorescence, Expressing, Incubation, Migration, Staining, Membrane
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