Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

doi: 10.1165/rcmb.2009-0135OC

Figure Lengend Snippet: Silver stained gel image of 100 μg of Cy3/Cy5 labeled protein obtained by lysis lavage from control mice separated in (A) pH range 4.5 to 5.5 or (B) pH range 5.5 to 6.7, depicting the location of (A) 44 or (B) 11 proteins identified by MALDI-TOF/TOF or LC/MS/MS.

Article Snippet: Cy3 and Cy5 maleimide monoreactive dyes and all other electrophoresis materials were obtained from GE Health Care (Piscataway, NJ).

Techniques: Staining, Labeling, Lysis, Liquid Chromatography with Mass Spectroscopy

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

doi: 10.1165/rcmb.2009-0135OC

Figure Lengend Snippet: Cy3/Cy5 labeling ratios for representative proteins showing significant differences in labeling ratios for NA (300 mg/kg) compared with DEM treatment. The bar graphs show labeling intensity of Cy3/Cy5 ratios along with Cy3 and Cy5 values for β-actin (A: spot #33), β-actin (B: spot #34), mitochondrial aldehyde dehydrogenase (C: spot #21), and Bip (D: spot #3). Values are the mean ± SD (n = 3).

Article Snippet: Cy3 and Cy5 maleimide monoreactive dyes and all other electrophoresis materials were obtained from GE Health Care (Piscataway, NJ).

Techniques: Labeling

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

doi: 10.1165/rcmb.2009-0135OC

Figure Lengend Snippet: Cy3/Cy5 labeling ratios for a mitochondrial aldehyde dehydrogenase protein, showing significant differences in labeling ratios for 200 mg/kg NA compared with 300 mg/kg NA. The bar graphs show labeling intensity of Cy3/Cy5 ratios along with Cy3 and Cy5 values for mitochondrial aldehyde dehydrogenase (spot #16). Values are the mean ± SD (n = 3).

Article Snippet: Cy3 and Cy5 maleimide monoreactive dyes and all other electrophoresis materials were obtained from GE Health Care (Piscataway, NJ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Single-Molecule Peptide Fingerprinting

doi: 10.1101/226712

Figure Lengend Snippet: (a) Substrates with two acceptor dyes were labeled at the N-terminal end and on cysteine residues ( Supplementary Table 1 ). (b) A typical time trace for Cy3 -NH 3 -17-C Cy5 -ssrA from three-color ALEX (top) showed FRET between Alexa488 and Cy3; and Alexa488 and Cy5 upon excitation with blue laser light (473 nm). Concurrent signals from Cy3 (middle) and Cy5 (bottom) upon direct excitation, respectively with green (532 nm) and red (637 nm). For clarity, an arbitrary offset of 200 a.u. was applied to the Alexa488 trace, and the sum of the Cy3 and Cy5 signals was plotted (middle). For the original trace, see Supplementary Figure 1e . (c) Comparison of acceptor dwell times. The dwell time of Cy5 (Δτ Cy5 ) was subtracted from that of Cy3 (Δτ Cy3 ) for each event from Cy3 -NH 3 -17-C Cy5 -ssrA (grey). The mean of the distribution is 3.5 ± 4.90 (sec). In white is the same analysis for Cy5 -NH 3 -17-C Cy3 -ssrA. The mean of the distribution is −3.9 ± 5.67 (sec). (d) Substrates labeled with one acceptor dye. Cysteine residues of the substrates (K-38-C-ssrA) were labeled with either Cy3 or Cy5. (e) A time trace from substrates in [d]. At time ~20 s, a Cy3-labeled substrate binds (top, Alexa488-Cy3 FRET) and 532 nm direct excitation (middle). At time ~50 s, a Cy5-labeled substrate bind (top, Alexa488-Cy5 FRET) and 637 nm direct excitation (bottom). An offset of 200 a.u. applied to Alexa488. (f) The percentage of processed Cy3-labeled substrates was plotted against the expected percentage of Cy3-labeled substrates. The line is a linear fit (slope of 0.98 ± 0.02, intercept 0.58 ± 0.79, R 2 = 0.99). Data points are from 100-s recordings, repeated ten times per condition (each n = 24.4 ± 1.50).

Article Snippet: ClpP was dialyzed overnight against PBS (pH 7.4) before labeling for 4 h at 4°C with monoreactive maleimide donor dye (Cy3, GE Healthcare, for two-color experiments, and Alexa488, Invitrogen, for three-color experiments).

Techniques: Labeling