protein band detection  (Bio-Rad)

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Full-length EGFR (gray) embedded in a nanodisc. The nanodisc is a lipid bilayer (beige) belted by an amphiphilic apolipoprotein (dark gray). EGFR consists of a 618-amino acid extracellular region that binds EGF (orange), a 27-amino acid transmembrane-spanning domain, and an intracellular region, which is a 37-amino acid juxtamembrane domain, a 273-amino acid kinase domain and a 231-amino acid disordered C-terminal tail. Green and maroon spheres indicate the donor and acceptor dyes, respectively . (b) Mean of zeta potential distributions for EGFR in nanodiscs containing increasing amounts of anionic lipids (0%, 15%, 30% and 60% POPS). Error bars are from three technical replicates. (c) Ensemble fluorescence emission spectra ( λ exc = 385 nm) of EGFR embedded Laurdan containing nanodiscs with increasing cholesterol. (d) Schematic of multiparametric single-molecule confocal microscope. (e) Fluorescence intensity for a representative image ( λ exc = 550 nm) where green spots are immobilized EGFR nanodiscs. (f) Representative fluorescence time trace from single-molecule FRET experiments showing number of detected photons for each 100 ms interval as intensity traces (green for donor; red for acceptor) with the average for each period of constant intensity (black solid line) and the corresponding donor lifetime (black dashed line).

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Zeta Potential Analyzer, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Cell-free reaction for the production of EGFR and nanodisc belt protein. Codon optimized DNA of the receptor protein (EGFR-SNAP) and the belt protein (ApoA1Δ49) are incubated overnight together with lipid vesicles with or without cholesterol and E. Coli lysate at 25 o C. (b) Stain-free (left) and fluorescence (right) gel images of the His-tag purified sample show the presence of ApoA1 at 25 kDa and full-length EGFR at 160 kDa, which implies successful EGFR production and insertion into nanodiscs. The presence of the EGFR band alone in the fluorescence gel image indicates successful and specific labeling. (c) Western blots were performed on labelled EGFR in nanodiscs. Anti-EGFR Western blots (left) and anti-phosphotyrosine Western blots (right) tested the presence of EGFR and its ability to undergo tyrosine phosphorylation, respectively, consistent with previous experiments on similar preparations. 1 , ,

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Incubation, Staining, Fluorescence, Purification, Labeling, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: Ensemble fluorescence excitation spectra ( λ em = 440 nm) of EGFR embedded Laurdan containing nanodiscs with 0% cholesterol, 7.5% cholesterol and 20% cholesterol. In the Laurdan excitation spectra, increase in the excitation band centered around 390 nm is observed with the addition of increasing amounts of cholesterol to the EGFR embedded nanodisc.

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Ensemble fluorescence excitation spectra ( λ em = 660 nm) of ss594 labeled EGFR nanodiscs with 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (b) Ensemble fluorescence emission spectra ( λ exc = 565 nm) of ss594 labeled EGFR nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (c) Ensemble time-correlated single photon counting measurements for ss594 labeled EGFR nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. The instrument response function (IRF) is shown in gray.

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Fluorescence, Labeling

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Ensemble fluorescence excitation spectra ( λ em = 700 nm) of cy5 labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (b) Ensemble fluorescence emission spectra ( λ ex = 630 nm) of cy5 labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS lipids. (c) Ensemble time-correlated single photon counting measurements were performed for cy5 labeled nanodiscs in 0% POPS, 15% POPS, 30% POPS and 60% POPS. The instrument response function (IRF) is shown in gray.

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Fluorescence, Labeling

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Confocal fluorescence image of immobilized constructs of EGFR in nanodiscs labeled with ss594 ( λ exc = 550 nm). (b) Representative intensity time trace from a single construct. The number of detected photons for each 100 ms interval was calculated and used to generate a fluorescence intensity trace (green) with the average intensity for the emissive period overlaid (black). (c) Histogram of the arrival times of detected photons generates the donor lifetime decay profile. Representative decay profiles of EGFR (green) with fit curve (black). The instrument response function (IRF) is shown in gray.

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Fluorescence, Construct, Labeling

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Confocal fluorescence image of immobilized constructs of EGFR in nanodiscs containing a labeled Cy5 lipid ( λ exc = 640 nm). (b) Representative intensity time trace from a single construct. The number of detected photons for each 100 ms interval was calculated and used to generate a fluorescence intensity trace (red) with the average intensity for the emissive period overlaid (black).

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Fluorescence, Construct, Labeling

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Fulllength EGFR in nanodiscs with partially anionic lipids (red). The negatively charged residues on the C-terminal tail are indicated in red and the positively charged residues on the kinase domain are indicated in blue. smFRET donor fluorescence lifetime distributions in (b) 100% DMPC, 0% POPS and (c) 100% POPC, 0% POPS; 85% POPC, 15% POPS; 70% POPC, 30% POPS; 40% POPC, 60% POPS without EGF (top); with 1 µ M EGF (bottom). (d) Full-length EGFR in nanodiscs with cholesterol (teal). (e) smFRET donor fluorescence lifetime distributions in 92.5% POPC, 7.5% cholesterol; 80% POPC, 20% cholesterol without EGF (top); with 1 µ M EGF (bottom). (f) Full-length EGFR in nanodiscs with cholesterol and anionic lipids. (g) smFRET donor fluorescence lifetime distributions in 62.5% POPC, 30% POPS, 7.5% cholesterol; 50% POPC, 30% POPS, 20% cholesterol without EGF (top); with 1 µ M EGF (bottom). Dotted lines indicate the maxima from a global fit of all lifetime distributions to a double Gaussian distribution model using maximum likelihood estimation. The maxima correspond to a compact and an open conformation with a distance of 8 nm and 12 nm, respectively, between the EGFR C-terminal tail and the membrane bilayer. (h) The amplitude of the open conformation (in %) in all the eight different membrane compositions in the absence (purple) and presence of EGF (orange). (i) The change in amplitude induced by EGF (black). The amplitude change upon EGF addition is high (22% – 55%) in 0% – 30% POPS but reduces drastically (0% – 6%) upon introduction of cholesterol in the lipid bilayer. The error bars in (h) and (i) are from the global fit.

Article Snippet: The fluorescence band detection was done using the ChemiDoc Imaging System (Bio-Rad Laboratories).

Techniques: Fluorescence, Membrane