alexa fluor 555 c2 maleimide  (Thermo Fisher)

Journal: Protein Science : A Publication of the Protein Society

Article Title: Fine‐tuned interactions between globular and disordered regions of single‐stranded DNA binding ( SSB ) protein are required for dynamic condensation under physiological conditions

doi: 10.1002/pro.70109

Figure Lengend Snippet: SSB condensate formation becomes crowder‐dependent in the absence of the conserved C‐terminal peptide (CTP). (a), (b) Epifluorescence microscopic images obtained upon mixing 15 μM SSB (protein concentration optimized based on Harami et al., ) and 0.15 μM Alexa555‐labeled SSB; (a) wild‐type (WT) SSB, hereinafter referred to as SSB; (b) SSBdC construct comprising amino acid residues (aa) 1–170, lacking the CTP in standard LLPS buffer complemented with polyethylene glycol (PEG 20,000, hereinafter referred to as PEG) at the indicated concentrations. Samples were incubated for the indicated times before imaging. (c), (d) PEG concentration dependence of the total droplet area of (c) SSB and (d) SSBdC fluorescence images from panels (a), (b), determined from the middle ROI (region of interest, see Methods). Means ± SEM are shown for n = 3. (e) PEG concentration dependence of turbidity ( OD 600 ) values recorded upon mixing 15 μM unlabeled SSB or SSBdC in standard LLPS buffer with indicated concentrations of PEG, followed by 1‐min incubation. Means ± SEM are shown for n = 3. Error bars are within symbols when not visible.

Article Snippet: SSB variant harboring the G26C aa substitution was labeled with FITC (Fluorescein 5‐isothiocyanate, Sigma‐Aldrich) or Alexa555 (Alexa Fluor 555 C2 Maleimide, Thermo Fisher) as in Harami et al. ( ).

Techniques: Protein Concentration, Labeling, Construct, Incubation, Imaging, Concentration Assay, Fluorescence

Journal: Protein Science : A Publication of the Protein Society

Article Title: Fine‐tuned interactions between globular and disordered regions of single‐stranded DNA binding ( SSB ) protein are required for dynamic condensation under physiological conditions

doi: 10.1002/pro.70109

Figure Lengend Snippet: ssDNA inhibits condensate formation by both SSB and SSBdC under permissive (crowded, glutamate‐free) conditions; SSB condensation becomes glutamate‐inhibited in the absence of the CTP. (a), (b) Epifluorescence microscopic images obtained upon mixing 30 μM SSBdC (protein concentration optimized based on Harami et al. ) and 0.3 μM Alexa555‐labeled SSBdC in standard LLPS buffer with indicated concentrations of 79‐mer homo‐deoxythymidine (dT 79 ) ssDNA, in the (a) absence and (b) presence of 150 mg/mL BSA as molecular crowder. Images were captured immediately or after 1‐h incubation as indicated and were not background corrected. (c) dT 79 concentration dependence of turbidity ( OD 600 ) values recorded upon mixing 15 μM unlabeled SSB and SSBdC and the indicated dT 79 concentrations in standard LLPS buffer containing 30 mg/mL PEG, followed by 1‐min incubation. Means ± SEM are shown for n = 3. (d), (e) Turbidity ( OD 600 ) values recorded upon mixing 10 μM unlabeled SSB or SSBdC in standard LLPS buffer containing (d) 150 mg/mL BSA or (e) 30 mg/mL PEG and the indicated concentrations of different salts, followed by 1‐min incubation. Means ± SD are shown for n = 3. Solid lines show best‐fits based on the Hill equation. See Table for half‐maximal effective concentration values.

Article Snippet: SSB variant harboring the G26C aa substitution was labeled with FITC (Fluorescein 5‐isothiocyanate, Sigma‐Aldrich) or Alexa555 (Alexa Fluor 555 C2 Maleimide, Thermo Fisher) as in Harami et al. ( ).

Techniques: Protein Concentration, Labeling, Incubation, Concentration Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Fine‐tuned interactions between globular and disordered regions of single‐stranded DNA binding ( SSB ) protein are required for dynamic condensation under physiological conditions

doi: 10.1002/pro.70109

Figure Lengend Snippet: CTP deletion slightly narrows the pH range for SSB condensate formation. (a) Epifluorescence microscopic images obtained upon mixing 15 μM SSB and 0.15 μM Alexa555‐labeled SSB (upper row) or 15 μM SSBdC and 0.15 μM Alexa555‐labeled SSBdC (lower row) in standard LLPS buffer complemented with 30 mg/mL PEG, buffered to the indicated pH values. The buffering agent was citrate, MES, and Tris, in pH ranges 3–4, 4.5–6.5, and 7–10, respectively (see also “Methods”). Samples were incubated for 1 h before imaging. (b) pH dependence of total droplet area of SSB and SSBdC condensates determined from the middle ROI of epifluorescence images of panel (a). Means ± SEM are shown for n = 3. (c) pH dependence of turbidity ( OD 600 ) values recorded upon mixing 15 μM unlabeled SSB or SSBdC in conditions described for panel (a), followed by 1‐min incubation. Means ± SEM are shown for n = 3.

Article Snippet: SSB variant harboring the G26C aa substitution was labeled with FITC (Fluorescein 5‐isothiocyanate, Sigma‐Aldrich) or Alexa555 (Alexa Fluor 555 C2 Maleimide, Thermo Fisher) as in Harami et al. ( ).

Techniques: Labeling, Incubation, Imaging

Journal: Protein Science : A Publication of the Protein Society

Article Title: Fine‐tuned interactions between globular and disordered regions of single‐stranded DNA binding ( SSB ) protein are required for dynamic condensation under physiological conditions

doi: 10.1002/pro.70109

Figure Lengend Snippet: SSB H55Y retains pH‐dependent condensation characteristic of wild‐type SSB. (a) Epifluorescence microscopic images obtained upon mixing 15 μM SSB and 0.15 μM Alexa555‐labeled SSBs (upper row: SSB, lower row: SSB H55Y ) at different pH values in buffers described for Figure , with the difference that PEG was not used in the current experiments. Samples were incubated for 1 h before imaging. (b) pH dependence of total droplet area of SSB and SSB H55Y condensates determined from the middle ROI of epifluorescence images of panels (a). Means ± SEM are shown for n = 3. (c) pH dependence of turbidity ( OD 600 ) values recorded upon mixing 15 μM unlabeled SSB or SSB H55Y in conditions described for panel (a), followed by 1‐min incubation. Means ± SEM are shown for n = 3. (d), (e) pH dependence of turbidity ( OD 600 ) values recorded for (d) unlabeled SSB and (e) SSB H55Y at the indicated concentrations in standard LLPS buffer as described for panel (a), after 1‐min incubation. Means ± SEM are shown for n = 3 (error bars are mostly within symbols).

Article Snippet: SSB variant harboring the G26C aa substitution was labeled with FITC (Fluorescein 5‐isothiocyanate, Sigma‐Aldrich) or Alexa555 (Alexa Fluor 555 C2 Maleimide, Thermo Fisher) as in Harami et al. ( ).

Techniques: Labeling, Incubation, Imaging