Steady-state spectrofluorometry

Several instruments are available to monitor the absorbance, emission or polarization characteristics of fluorescent molecules over time or under special environmental conditions (low temperature or high pressure).

Spectrophotometer Fluorolog

The Fluorolog 3.22 (Jobin Yvon-Spex) (manual) is the "work-horse" of the lab because of its versatility: It can acquire excitation- and emission spectra (example: BFP, CFP, YFP, GFP, DsRed) (2D-fluorescence), polariza-tion (anisotropy), chemiluminescence and monitor time-dependent reactions. It is also capable of handling solid samples, microtiter plates and work with cryo- and high pressure-accessories.

Spectrophotometer Cary Eclipse

The Cary Eclipse (Varian is another heavily used spectrophotometer in our centre. It is possible to measure fluorescence using multi-well set up. Bleaching of the samples is strongly surpressed  due to the pulsed excitation light source used.

Publications

• Bollen, Y.J.M., Westphal, A.H., Lindhoud, S., Berkel, W.J.H. van & Mierlo, C.P.M. van (2012). Distant residues mediate picomolar binding affinity of a protein cofactor. Nature Communications, 3.
• Lindhoud, S., Westphal, A.H., Borst, J.W. & Mierlo, C.P.M. van (2012). Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein. PLoS One, 7(9).
• Malikova, N.P., Visser, N.V., Hoek, A. van, Skakun, V.V., Vysotski, E.S., Lee, J. & Visser, A.J.W.G. (2011). Green-Fluorescent Protein from the Bioluminescent Jellyfish Clytia gregaria Is an Obligate Dimer and Does Not Form a Stable Complex with the Ca2+-Discharged Photoprotein Clytin. Biochemistry, 50(20), 4232-4241.
• Visser, A.J.W.G., Vysotski, E.S. & Lee, J. (2011). Critical Transfer Distance Determination Between FRET Pairs. Photobiological Sciences Online.
• Visser, A.J.W.G. & Rolinski, O.J. (2010). Basic Photophysics. Photobiological Sciences Online.
• Borst, J.W., Willemse, M., Slijkhuis, R., Krogt, G. van der, Laptenok, S., Jalink, K., Wieringa, B. & Fransen, J.A.M. (2010). ATP Changes the Fluorescence Lifetime of Cyan Fluorescent protein via an Interaction with His148. PLoS One, 5(11), e13862.
• Visser, N.V., Westphal, A.H., Nabuurs, S.M., Hoek, A. van, Mierlo, C.P.M. van, Visser, A.J.W.G., Broos, J. & Amerongen, H. van (2009). 5-Fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin. FEBS Letters, 583(17), 2785-2788.
• Eremeeva, E., Markova, S.V., Frank, L.A., Lee, J., Berkel, W.J.H. van, Visser, A.J.W.G. & Vysotski, E.S. (2008). The kinetics of coelenterazine binding with apo-obelin and apo-aequorin. Luminescence, 23(2), 66-67.
• Koehorst, R.B.M., Spruijt, R.B. & Hemminga, M.A. (2008). Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment. Biophysical Journal, 94, 3945-3955.
• Nazarov, P.V., Koehorst, R.B.M., Vos, W.L., Apanasovich, V.V. & Hemminga, M.A. (2007). FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein. Biophysical Journal, 92(4), 1296-1305.
• Nazarov, P.V., Koehorst, R.B.M., Vos, W.L., Apanasovich, V.V. & Hemminga, M.A. (2006). FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association. Biophysical Journal, 91(2), 454-466.
• Slootweg, E.J., Keller, H.J.H.G., Hink, M.A., Borst, J.W., Bakker, J. & Schots, A. (2006). Fluorescent T7 display phages obtained by translational frameshift. Nucleic Acids Research, 34(20), e137.
• Borst, J.W., Hink, M.A., Hoek, A. van & Visser, A.J.W.G. (2005). Effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins. Journal of Fluorescence, 15(2), 153-160.
• Hesselink, R.W., Koehorst, R.B.M., Nazarov, P.V. & Hemminga, M.A. (2005). Membrane-bound peptides mimicking transmembrane Vph1p helix 7 of yeast V-ATPase: A spectroscopic and polarity mismatch study. Biochimica et Biophysica Acta. Biomembranes, 1716(2), 137-145.
• Visser, N.V., Borst, J.W., Hink, M.A., Hoek, A. van & Visser, A.J.W.G. (2005). Direct observation of resonance tryptophan-to-chromophore energy transfer in visible fluorescent proteins. Biophysical Chemistry, 116(3), 207-212.
• Koehorst, R.B.M., Spruijt, R.B., Vergeldt, F.J. & Hemminga, M.A. (2004). Lipid bilayer topology of the transmembrane alpha-helix of M13 major coat protein and bilayer polarity profile by site-directed fluorescence spectroscopy. Biophysical Journal, 87(3), 1445-1455.
• Hink, M.A., Visser, N.V., Borst, J.W., Hoek, A. van & Visser, A.J.W.G. (2003). Practical use of corrected fluorescence excitation and emission spectra of fluorescent proteins in Förster Resonance Energy Transfer (FRET) studies. Journal of Fluorescence, 13, 185-188.
• Verkhusha, V.V., Pozhitov, A.E., Smirnov, S.A., Borst, J.W., Hoek, A. van, Klyachko, N.L., Levashov, V. & Visser, A.J.W.G. (2003). Effect of high pressure and reversed micelles on the fluorescent proteins. Biochimica et Biophysica Acta. General subjects, 1622, 192-195.

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