cy5 maleimide mono reactive dye  (GE Healthcare)

Journal: Biomolecules

Article Title: Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly

doi: 10.3390/biom13060951

Figure Lengend Snippet: Single-molecule system to study the association of S4 in the presence of other RPs. ( a ) Nomura 30S ribosome assembly map highlighting RPs used in this study. ( b ) RPs surrounding the S4 binding site in the mature 30S ribosome. The 5WJ recognized by S4 is shown as a dark grey ribbon. PDB: 4V4A. ( c ) Schematic of the single-molecule system used to study the binding dynamics of S4 during and after transcription of the pre-16S rRNA. Green star, Cy3; red star, Cy5. ( d , e ) Sample raw single-molecule traces illustrating PIFE at the end of transcription (green; top) and colocalization of S4-Cy5 with the transcript (red; bottom). A rastergram for each transcript is shown below each plot of Cy5 intensity. This simplified annotation is used to visualize the timing of S4-Cy5 binding (black bars). PIFE (green circle) indicates the end of transcription. See Methods for details. ( f , g ) Rastergrams for 50 randomly selected pre-16S transcripts during and after transcription in the presence of ( f ) 100 nM unlabeled S12 and ( g ) 20 nM unlabeled S8. See for data with other RPs.

Article Snippet: Cy5-maleimide mono-reactive dye (GE-Healthcare, Chicago, IL, USA) was dissolved in DMSO to 20 mM and immediately added to the protein solution.

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly

doi: 10.3390/biom13060951

Figure Lengend Snippet: Stable binding of S4 is enhanced by S12. ( a ) Box plot of the distribution of S4-Cy5 dwell times in the presence of no other protein (–), 100 nM S12, 20 nM S8, 100 nM S5, and 50 nM S16. (****, p ≤ 0.0001; *, p ≤ 0.05; ns, p > 0.05; Student’s t -test) ( b ) Maximum likelihood analysis of the unbinned dwell time distribution shown in ( a ). Triple-exponential fit is shown with a colored line, and fitting parameters are reported in . Centers of the binned dwell time data for S12, S8, and –RPs are shown as circles, diamonds, and squares, respectively. See for additional data. ( c ) Fraction of pre-16S TECs that experienced an S4 binding event lasting longer than 20 s, for experiments as in ( a ). For comparison, the grey bar (+6) indicates the effect of adding six RPs at the same time: 20 nM S20, 20 nM S17, 20 nM S8, 50 nM S16, 100 nM S5, 100 nM S12; data from Ref. . Bars, average; grey circles, individual replicates. See for the number of transcripts analyzed in each experiment.

Article Snippet: Cy5-maleimide mono-reactive dye (GE-Healthcare, Chicago, IL, USA) was dissolved in DMSO to 20 mM and immediately added to the protein solution.

Techniques: Binding Assay

Journal: Biomolecules

Article Title: Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly

doi: 10.3390/biom13060951

Figure Lengend Snippet: S12-Cy5 only interacts transiently with pre-16S transcripts in the absence of other RPs. ( a ) Single-molecule assay to measure S12 binding during transcription. ( b ) Example of a single-molecule trace illustrating transcription from a single Cy3-TEC (green) and colocalization of S12-Cy5 (red). ( c ) Rastergram of S12-Cy5 binding (black bars) to 50 randomly selected pre-16S TECs, as in . ( d ) MLE analysis of S12-Cy5 binding to pre-16S transcripts (red) compared to S4-Cy5 (gold). The characteristic S12-Cy5 dwell time is τ = 0.46 s; see for S4-Cy5 parameters. ( e ) The fraction of pre-16S TECs with stable S4-Cy5 binding is dependent on S12 concentration, as 20 nM unlabeled S12 does not improve S4-Cy5 binding.

Article Snippet: Cy5-maleimide mono-reactive dye (GE-Healthcare, Chicago, IL, USA) was dissolved in DMSO to 20 mM and immediately added to the protein solution.

Techniques: Binding Assay, Concentration Assay

Journal: Biomolecules

Article Title: Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly

doi: 10.3390/biom13060951

Figure Lengend Snippet: S12 is more effective when present during transcription. Strategy for visualizing S4-Cy5 binding to the pre-16S after transcription. Immobilized transcripts were detected by hybridization of a complementary Cy3-oligomer. ( a ) Binding to pre-16S transcribed in the presence of 100 nM S12. Rastergram of S4-Cy5 binding at right. Pre-16S transcripts were ordered by the start of the first S4-Cy5 binding event longer than 20 s. ( b ) Binding in the presence of 100 nM S12, as in ( a ). ( c ) Binding in the absence of S12, as in ( a ). See for binding lifetimes.

Article Snippet: Cy5-maleimide mono-reactive dye (GE-Healthcare, Chicago, IL, USA) was dissolved in DMSO to 20 mM and immediately added to the protein solution.

Techniques: Binding Assay, Hybridization

Journal: Biomolecules

Article Title: Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly

doi: 10.3390/biom13060951

Figure Lengend Snippet: The presence of S12 influences S4 binding during transcription and after transcription. ( a ) Fraction of pre-16S rRNAs that bind S4 > 20 s in the absence of S12 (black bar; as in c), in the presence of S12 after transcription (red bar; as in b), and in the presence of S12 during transcription (teal bar; as in a). Gray symbols indicate the values for independent replicates. ( b ) Cumulative probability plot of S4-Cy5 arrival times for specific events lasting >1 s. Association times were combined from two independent replicates. Association of S4-Cy5 is faster in the presence of S12 added during or after transcription. The cumulative density functions for S12 co-txn and S12 post-txn are statistically similar, and both are statistically different than no S12 (K–S test). ( c ) Cumulative probability plot of S4-Cy5 arrival times for stable events lasting >20 s. Apparent association times were fit with a single exponential function (lines). Stable association of S4-Cy5 is enhanced by the presence of S12 during and after transcription.

Article Snippet: Cy5-maleimide mono-reactive dye (GE-Healthcare, Chicago, IL, USA) was dissolved in DMSO to 20 mM and immediately added to the protein solution.

Techniques: Binding Assay

Journal: Advanced Science

Article Title: Remediation of Metal Oxide Nanotoxicity with a Functional Amyloid

doi: 10.1002/advs.202310314

Figure Lengend Snippet: Rescue of HUVEC early/late cell apoptosis and cell necrosis induced by CuO and ZnO nanoparticles with bLg amyloid. A) Flow cytometry was used to classify necrotic and apoptotic HUVECs upon 24 h of exposure to CuO or ZnO nanoparticles (100, 150 µ m ) in the presence and absence of the bLg amyloid (5 mg mL −1 ). Apoptotic cells were labeled with Annexin V‐FITC, and necrotic cells were labeled with propidium iodide (Q3‐1: necrosis. Q3‐2: late apoptosis. Q3‐3: normal cells. Q3‐4: early apoptosis). B) Statistical evaluation of normal, apoptotic, and necrotic (%) cells in the control and treated cell groups presented in panel (A). Data were analyzed via two‐way ANOVA followed by Tukey's post‐hoc test for multiple comparisons (n = 3). C) Adhesion rate of the bLg amyloid on the surface of HUVECs in the presence and absence of endocytosis inhibitors. HUVECs were treated with MβCD (5 m m for 2 h) and MDC (10 µ m for 1 h) before the bLg treatment. HUVECs were exposed to the bLg amyloid labeled with Cy5 (Cy5‐bLg) for 4 h, and Cy5 positive cells were detected by flow cytometry. D) Statistical analysis of Cy5 positive cells derived from the assay presented in panel (C). Data were analyzed via one‐way ANOVA followed by Tukey's post‐hoc test for multiple comparisons (n = 3). E) Confocal fluorescence images depicting the localization of the Cy5‐bLg amyloid (2.5 mg mL −1 ) in non‐/pre‐treated (MβCD/MDC) HUVECs upon exposure for 4 h. Channels: Nuclei (blue), Cy5‐bLg amyloid (purple), F‐actin (green). Scale bars: 20 µm, n = 3. All the data are depicted as mean ± SD. Significance between the treated and control groups is denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. Similarly, # p < 0.05, ## p < 0.01, and ### p < 0.001 represented the significant differences between two treated groups.

Article Snippet: For the synthesis of cyanine5‐labeled bLg (Cy5‐bLg) amyloid, 10 mg of the mono‐reactive succinimidyl dye ester of Cy5 (NHS‐Cy5, Pierce, USA) was completely dissolved in 1 mL of dimethyl sulfoxide.

Techniques: Flow Cytometry, Labeling, Control, Derivative Assay, Fluorescence

Journal: bioRxiv

Article Title: SERF2, an RNA G-quadruplex Binding Protein, promotes stress granule formation

doi: 10.1101/2023.10.09.561572

Figure Lengend Snippet: (A) Phase regimes illustrating LLPS in SERF2 as a function of total RNA concentration extracted from HEK293T cells. (B) Fluorescence imaging shows gel-like structures in 50 µM SERF2 (left) mixed with 200 ng/µL of total RNA containing 10% (w/v) PEG8000 incubated for 30 minutes at room temperature. ( C-E ) 50 µM of SERF2 dissolved in 20 mM NaPi (pH 7.4), 100 mM KCl readily undergoes LLPS formation when mixed with equimolar concentration of rG4 quadruplexes that include TERRA, (G4C2) 4 , and (UG4U) 6 . The sample mixture contains 1/200 th Cy-5 label protein (purple signals) and 6-FAM rG4 quadruplex (green signals) as indicated in the figure inset. Two-component FRAP analysis are done to measure the protein recovery (purple plot) and rG4 quadruplex recovery (green plot) rates in the SERF2-rG4s droplets. The pre-bleached, after-bleached (0 s) and recovered droplets (300 s) are shown on the top of each FRAP plot. The FRAP data were fitted in GraphPad Prism using non-linear regression one-phase association model to obtain the recovery halftime (t1/2). (F,G) Schematics showing SERF2 and TERRA sample mixture phase separation in 10% PEG8000 at different protein to RNA concentrations (F), and at different salt and PEG8000 concentrations (G) . The black and purple spheres in all phase regime represents no formation or formation of droplets, respectively. (H) Dynamics and recovery of Cy5-labelled proteins in SERF2-total RNA droplets obtained by FRAP analysis suggest the gel-like structures are dynamic and reversible. Standard errors are calculated by analyzing 8 isolated droplets subjected to FRAP. (I) DIC and Fluorescence images showing co-phase separation of SERF2 (purple) and G3BP1 with HeLa total RNA. (J) SERF2 facilitates G3BP1-RNA condensation. 0, 25 or 50 µM of SERF2 protein was added to 12.5 ng/µl of HeLa total RNA with or without 25 µM of G3BP1.

Article Snippet: Cy-5 labeling was done by incubating 200 μM of SERF2 (T2C) with a 10-molar excess of Cy5 maleimide mono-reactive dye (Cytiva, PA25031) in 20 mM Tris-HCl pH7.4 and 100 mM KCl buffer overnight, at 25 °C, under continuous shaking at 300 rpm.

Techniques: Concentration Assay, Fluorescence, Imaging, Incubation, Isolation