PMC

Time‐resolved emission intensity (λ _ex=292–366 nm, integration time=60–400 μs) of Tb^III emission (λ _em=510–550 nm) and AF633 emission (λ _em=660–670 nm). The tagging reaction was performed by incubating 15 μM aurora A with 120 mM Tb.C12 and different amounts of AF633 at 4 °C for 18 h in 50 mM Tris⋅HCl, 50 mM NaCl, 1.8 % DMSO at pH 7.4. Control experiments involved aurora A with no Tb^III complex (red) or only AF633 (orange). a) Tb^III emission after incubation of aurora A with different equivalents of AF633 (relative to aurora A). b) Same as (a), but monitoring AF633 emission. Observation of some background emission without AF633 is expected from the emission spectrum of the Tb^III tag (Figure a). c) Tb^III emission lifetimes (λ _ex=280 nm, λ _em=545 nm) of the samples in (b).

Activation properties of CFDA-Tz. (a) Activation of CFDA-Tz with various amounts of cell lysate. CFDA-Tz dye (20 µM) was incubated at room temperature with HT1080 cell lysate (0, 1, 2.5, 5, 10 or 20 µg). Fluorescence emission was recorded at 526 nm over time; (b) Emission spectrum of CFDA-Tz before and after activation by lysate. CFDA-Tz dye (20 µM) was incubated with HT1080 cell lysate (0 or 10 µg) for 40 min at room temperature. The fluorescence emission scan was then recorded.

The measured spectral irradiance of the sky at mid-twilight (solar altitude −7.2°). This is close to the maximum effect of ozone absorption on the colour of the downwelling irradiance from the scattering of sunlight by the illuminated atmosphere within and above the ozone layer. The dashed lines show thermal (Planck) spectra with temperatures of 5800 K (approx. sunlight) and 20 000 K. The narrow emission peak at 590 nm results from the resonance scattering of solar photons in the sodium D-lines from atoms coming from ablated meteors that form a layer between 80 and 105 km altitude, above the ozone layer. The strong, broad absorption bands at the red end of the spectrum are from O₂ and H₂O in the atmosphere. At high latitudes, the aurora can radiate strong emission lines, principally at 558 and 630 nm, from neutral oxygen atoms above an altitude of around 100 km. (Online version in colour.)

(A) Primary amino acid structure of FRET reporters: full-length MCAK (FMCAK; dimer), MCAK without the NT (FMΔNT; dimer), MCAK without the CT (FMΔCT; monomer), or FRET positive control protein (FCP). Primary amino acid structure of GFP fusion proteins: MCAK without the NT (GMΔNT), MCAK without the CT (GMΔCT), MCAK with the neck and catalytic domain (GMD), or the MCAK catalytic domain alone (GCD). The CT fused to GST is also shown (GST-CT). Scale bar, 100 amino acids.
(B, C) Purified protein was excited at 433 nm, the emission spectra recorded from 445–600 nm, and the average emission spectrum graphed from three independent experiments.
(D) 10% SDS-PAGE Coomassie stained gel of the input and bound fractions of a representative glutathione pull-down assay in which GST-CT or GST alone were incubated with control GFP, GMΔCT, GMD, or GCD. MW, molecular weight markers. Input, protein input.
(E) Quantification of three independent pull-down experiments. Average pmol ± SEM of MCAK proteins bound to GST-CT is graphed.