User manual LEICA CONFOCAL APPLICATION LETTER BROCHURE 02-2007

[. . . ] They can be detected either by membrane permeable ligands attached to fluorescent dyes bearing a high affinity to the protein motive or ligands which undergo a covalent binding with the modified enzyme. The HaloTag Interchangeable Technology (Promega, Mannheim, Germany) is based on the genetic mutation of a monomeric prokaryotic hydrolase enzyme expressed Table 1: Hardware settings of Leica TCS SP5 laserscan microscope HaloTag® diAcFAM HCX PL APO 63. 0x1. 20 WATER UV 1400 Hz bidirectional scan 488 nm 495­600 nm 256 x 256 pixels ~6 ~100 % 5% ­ 100 % ­ circle (diameter 3 µm) 10 x 118 ms 1 x 118 ms 50 x 118 ms 1s HaloTag® TMR HCX PL APO 63. 0x1. 20 WATER UV 1400 Hz bidirectional scan 561 nm 568­672 nm 256 x 256 pixels ~6 ­ 0% 10 % 0% 100 % circle (diameter 3 µm) 10 x 118 ms 1 x 118 ms 50 x 118 ms 1s * First published in: Biospektrum, 05 Sep 2006, pp. 515-517 Objective Scan speed (Line frequency) Excitation wavelength Emission range Format Zoom Laser power (Argon laser) AOTF (imaging) 488 nm AOTF (imaging) 561 nm AOTF (bleaching) 488 nm AOTF (bleaching) 561 nm ROI geometry Prebleach Bleach Postbleach 1 Postbleach 2 30 x 2 Confocal Application Letter FRAP in the cells as a monomer. Due to its prokaryotic origin, endogenous activities in mammalian cell systems can almost be excluded. [. . . ] Cells stained with the HaloTag-diAcFAM ligand showed a decrease in fluorescence by 35% of the original intensity after the bleaching pulse. 30 seconds later, the original intensity was reached (Fig. This experiment represents other experiments with HaloTag-diAcFAM stained cells. In all experiments, the half-time recovery of 1. 14 +/- 0. 38 seconds was observed. Cells stained with HaloTag TMR ligand were inves-tigated under the same conditions recovered with a half-time of 1. 24 +/- 0. 15 seconds, a slightly slower kinetic (Fig. For image acquisition before and after bleaching, the DPSS 561nm laser was used. To achieve more effective bleaching, the 488 nm Argon laser was additionally applied only for A B C D E F Figure 1: A­C, -Tubulin-HaloTag fusion protein stained with HaloTag-diAcFAM ligand. D-F, p65-HaloTag fusion protein stained with HaloTag-TMR ligand. (A, D) fluorescence, (B, E) transmitted light, (C, F) Overlay of fluorescence- and transmitted light image. A B 1. 1 1 0. 9 0. 8 0. 7 0. 6 0. 5 0. 4 0. 3 0. 1 0 0 5 10 15 20 25 30 35 [s] (1) (3) (2) Figure 2: A, FRAP experiment with -Tubulin-HaloTag fusion protein stained with HaloTag-diAcFAM-Ligand. B, 1) last prebleach, 2) bleach pulse with 488 nm argon-laser line, 3) last postbleach. Confocal Application Letter 3 FRAP Leica Microsystems Heidelberg GmbH · Am Friedensplatz 3 · D-68165 Mannheim, Germany · Tel. +49 (0) 6 21-70 28-0 · E-Mail: clsm. support@leica-microsystems. com LEICA and the Leica Logo are registered trademarks of Leica IR GmbH. Here the initial fluorescence intensity decreased more than 80% and after 30 seconds the fluorescence reached 85 % of the initial fluorescence (Fig. Non-transfected cells were as control of the specificity of the staining with both ligands. With the excitation wavelengths mentioned above the cells did not show any detectable fluorescence (data not shown) either for diAcFAM or for TMR. Discussion The use of new specific fluorescence ligands[5-7] with different spectral characteristics allows for more flexibility to detect non-fluorescent proteins. Timeconsuming and cumbersome sub-cloning work can be avoided with the described methods. However, potential toxicity and specificity need always to be taken into account when applying these technologies. Since the HaloTag Interchangeable Technology is based on a prokaryotic protein, non-specific staining in mammalian cells is significantly reduced. Additionally, toxicity could not be observed during longterm investigations[7]. We used the Leica TCS SP5 confocal microscope for the FRAP analysis of human -Tubulin and p65-HaloTag fusion proteins. [. . . ] [3] Lippincott-Schwartz, J. , Snapp, E. , Kenworthy, A. (2001): Studying protein dynamics in living cells. (2001): From fixed to FRAP: measuring protein mobility and activity in living cells. (1998): Fluorescent labeling of recombinant proteins in living cells with FlAsH. [. . . ]