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: Detection of in‐gel fluorescence (IGF) from endogenously expressed proteins tagged with yeGFP. (a) Control (WT: wild‐type) yeast or yeast expressing Bmh1‐yeGFP and Hxk1‐yeGFP were lysed in the indicated conditions, samples were resuspended in LDS sample buffer (1X final, Bio‐Rad) 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 green fluorescence using a Typhoon and a Chemidoc MP, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. After transfer to a nitrocellulose membrane, proteins were immunoblotted with anti‐GFP antibodies (Roche) and detected by chemiluminescence using anti‐mouse antibodies coupled to HRP. (b) Protein lysates were prepared as in (a). Proteins were incubated at 30°C or 95°C for 5 min, with and without prior incubation at 30°C (5 min). After migration on a commercial precast 4%–20% TGX gel (Bio‐Rad), gels were imaged for green fluorescence using 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 imaged again for yeGFP fluorescence, which was maintained during transfer. Proteins were then immunoblotted with anti‐GFP antibodies (Roche) and anti‐mouse antibodies coupled to Alexa Fluor 680. yeGFP fluorescence was then visualized again before detecting the anti‐GFP antibodies by fluorescence on a Chemidoc MP. GFP, green fluorescent protein; HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate; Nat., native conditions; TCA, lysis in denaturing conditions with TCA.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Fluorescence, Control, Expressing, Incubation, Migration, Staining, Membrane, Lysis

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 yeGFP‐, GFP(S65T)‐, and EGFP‐tagged proteins. (a). Yeast expressing Bmh1‐yeGFP, Bmh1‐GFP(S65T), Hxk1‐yeGFP, and Hxk1‐GFP(S65T) 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 gels (Bio‐Rad), gels were imaged for green fluorescence using 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 imaged again for green fluorescence, which was maintained during transfer. Proteins were then immunoblotted with anti‐GFP antibodies (Roche) and anti‐mouse antibodies coupled to Alexa Fluor 680. Green fluorescence was then visualized again before detecting the anti‐GFP antibodies by fluorescence. The membranes were then stripped and incubated with anti‐Bmh1 or anti‐Hxk1/2 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. (b) Yeast expressing Bmh1‐yeGFP and Bmh1‐EGFP 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 green 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. GFP, green fluorescent protein; HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Comparison, Fluorescence, Expressing, Incubation, Migration, Staining, Membrane

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 yeGFP‐, sfGFP‐, and mNeonGreen‐tagged proteins. Yeast expressing Bmh1‐yeGFP, Bmh1‐sfGFP, or Bmh1‐mNeonGreen 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 green 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. HRP, horseradish peroxidase; sfGFP, superfolder green fluorescent protein.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Comparison, Fluorescence, Expressing, Incubation, Migration, Staining, Membrane

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: Temperature‐sensitivity of in‐gel fluorescence (IGF) of yeGFP‐, EGFP‐, and sfGFP‐tagged proteins. (a) Yeast expressing Bmh1‐yeGFP 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 (note that the temperature range is different for sfGFP). After migration on a commercial precast 4%–20% TGX gel (Bio‐Rad), gels were imaged for green fluorescence using a Typhoon, 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. (b) Same as (a) using yeast expressing Bmh1‐EGFP. (c) Same as (a) using yeast expressing Bmh1‐sfGFP. (d) Quantification of the green fluorescence signal as a function of the denaturation temperature for various green FP‐tagged Bmh1 ( n = 3; ±SD). Solid line: sigmoidal fit of the data (GraphPad Prism), dotted line: 95% confidence interval of the fit. FP, fluorescent proteins; GFP, green fluorescent protein; HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate; sfGFP, superfolder green fluorescent protein.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Fluorescence, Expressing, Incubation, Migration, Staining, Membrane

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: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Comparison, Fluorescence, Expressing, Incubation, Migration, Staining, Membrane

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: Sensitivity and linearity of in‐gel fluorescence (IGF) detection of EGFP. (a) (Left panel) Yeast expressing Bmh1‐EGFP were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at 30°C. Samples were serially diluted (right to left) into sample buffer without LDS (see Material and Methods), and loaded onto on a commercial precast 4%–20% TGX gel (Bio‐Rad). After migration, gels were imaged for green fluorescence using a Typhoon, and 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. (Right panel) Quantification of the signals obtained for green fluorescence (black) and chemiluminescence (red) as a function of sample dilution. Linear‐scaled and log‐plot scaled graphs are shown. The regression coefficient of a linear fitting is indicated. (b) (Left panel) Same experiment as in (a) but using anti‐GFP antibodies and anti‐mouse antibodies coupled to HRP to reveal proteins by chemiluminescence. (Right panel). Quantification of the signals obtained for fluorescence (black) and chemiluminescence (red) as a function of sample dilution. Linear‐scaled and log‐plot scaled graphs are shown. The regression coefficient of a linear fitting is indicated. HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Fluorescence, Expressing, Incubation, Migration, Membrane

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: Detection of endogenous yeast EGFP‐tagged proteins with various expression levels and correlation with published abundances. (a) Proteins tagged with EGFP for in‐gel fluorescence (IGF) detection. Abundance (median) is according to Ho et al. (2018) and available at www.yeastgenome.org . (b) Protein lysates prepared in native conditions from yeast expressing the indicated proteins fused to EGFP samples were resuspended in LDS sample buffer and incubated for 5 min at the 30°C and loaded onto on a commercial precast 4%–20% TGX gel (Bio‐Rad). After migration, gels were imaged for green fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. A representative gel is shown. ER, endoplasmic reticulum; PM, plasma membrane. (c) Correlation of IGF intensities with published abundance based on quantitative mass spectrometry experiments, western blot experiments, or confocal microscopy experiments. Data were from Ho et al. (2018) and retrieved from SGD ( www.yeastgenome.org ) for untreated/unchallenged cells.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Expressing, Fluorescence, Incubation, Migration, Staining, Clinical Proteomics, Membrane, Mass Spectrometry, Western Blot, Confocal Microscopy

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: In‐gel fluorescence (IGF) detection in the context of co‐immunoprecipitation or SNAP‐tagging. (a) Protein lysates prepared in native conditions with native immunoprecipitates (IP) lysis buffer from yeast expressing Bmh2‐ymCherry (a yeast codon‐optimized mCherry) with or without the co‐expression of Bmh1‐yeGFP were subjected to co‐immunoprecipitation using GFP‐trap beads. IP were incubated at 50°C for 20 min in 1X Laemmli sample buffer at pH 8.0 which allows to maintain protein fluorescence at low protein concentrations. Input samples and IP are shown. After migration on a commercial precast 4%–20%TGX gel (Bio‐Rad), gels were imaged for green and red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. * indicates bleed‐through of the green fluorescence into the red channel. (b) Protein lysates prepared in native conditions from yeast expressing Bmh1‐yeGFP with or without the co‐expression of Bmh2‐ymCh were subjected to co‐immunoprecipitation using RFP‐trap beads and treated as in (a). Input samples and IP are shown. After migration on a commercial precast 4%–20% TGX gel (Bio‐Rad), gels were imaged for green and red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. (c) Protein lysates prepared in native conditions with native IP lysis buffer from yeast expressing Reg1‐SNAP and Snf4‐mCherry, and were treated or not with SNAP‐surface Alexa Fluor 647. After migration on a commercial precast 4%–20% TGX gel (Bio‐Rad), gels were imaged for red and far‐red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc MP.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Fluorescence, Immunoprecipitation, Lysis, Expressing, Incubation, Migration, Staining

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: In‐gel fluorescence (IGF) detection in cells from various organisms. (a) Drosophila wing‐imaginal‐disc cultured cells (Clone 8 [Cl‐8] cells) were transfected with plasmids encoding the Hedgehog (HH) transducer Smoothened (SMO) fused to EGFP and EGFP alone. They were also transfected (+) or not (−) with a construct allowing the expression of HH. Cells were lysed in native conditions (see Material and Methods), lysates were mixed with 4X Laemmli sample buffer and incubated for 5 min at 25°C before loading on a homemade PAGE gel. IGF was detected on a Typhoon. SMO‐EGFP is phosphorylated in the presence of HH, causing a slower migration (red arrowhead). (b) Transgenic Drosophila flies expressing or not an EGFP‐tagged version of the polarity protein PAR‐3 were dissected and ovaries were lysed in native conditions (see Material and Methods). Samples were incubated for 5 min at 72°C in LDS sample buffer, loaded on a NuPage Bis‐Tris gel (ThermoFisher) and IGF was detected on a Typhoon after migration. After fluorescence imaging, total proteins were stained with Coomassie stain and imaged on the infrared channel on a Chemidoc MP. (c) MDCK cells expressing Arf1 fused to EGFP or mCherry were lysed in native conditions (see Material and Methods). Samples were incubated for 10 min at 50°C in LDS sample buffer and loaded on a gel. After migration on a commercial precast 4%–20%TGX gel (Bio‐Rad), gels were imaged for green and red fluorescence using a Chemidoc MP, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. Note that some bleed‐through of green fluorescence is observed in the red channel (see also Figure and ). (d) Transgenic C. elegans expressing or not GFP‐tagged alpha‐tubulin and mCherry‐tagged histone H2B were lysed in native conditions (see Material and Methods) by sonication. Samples were incubated for 5 min at 30°C or 50°C in Laemmli sample buffer (pH 8.0). After migration on a commercial precast 4%–20% TGX gel (Bio‐Rad), gels were imaged for green and red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. * denotes a non‐specific fluorescence protein present in extracts of wild‐type C. elegans whose fluorescence disappears when heating the sample at 50°C. LDS, lithium dodecyl sulfate.

Article Snippet: Total proteins were visualized by IGF using a trihalo compound incorporated in the commercial pre‐cast SDS–PAGE gels (stain‐free TGX gels, 4%–20%; Bio‐Rad) after 45 s UV‐induced photoactivation using a ChemiDoc MP imager (Bio‐Rad), serving as a loading control.

Techniques: Fluorescence, Cell Culture, Transfection, Construct, Expressing, Incubation, Migration, Transgenic Assay, Imaging, Staining, Sonication