Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus
裂殖酵母细胞核的荧光光漂白技术（FRAP）
发布: 2013年10月20日第3卷第20期 DOI: 10.21769/BioProtoc.941 浏览次数: 10559
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
We use fluorescence recovery after photobleaching (FRAP) to calculate the diffusion coefficient of GFP in the nucleoplasm of fission yeast. The FRAP method can be generally used to measure the mobility of proteins inside the cell or its organelles.
In our experiment we only measured the diffusion of GFP inside the nucleoplasm of fission yeast mitotic cells. However, if GFP is fused to a protein, the mobility of the protein of interest can be calculated following the GFP signal in the bleached area. We did not, however, address this in our experiments; therefore other sources could be searched for this topic.
To compare FRAP and FLIP, both techniques can be used to measure protein mobility inside a cell. However, with FRAP, the diffusion of a protein is measured in the region of interest (ROI), to observe the recovering of fluorescence in this area. In FLIP, fluorescence recovery is measured in an area different from where the bleaching was done, to observe whether the tagged protein is able to move into that area, which would become darker, gaining the bleached proteins. The major difference here is that for FRAP a single bleaching event is sufficient, while FLIP requires a number of bleaching steps, in order to avoid reflux of fluorescent protein in the same region.
Technically FRAP in the nucleus und FRAP in the cytoplasm has no difference. However, we measured a difference between the diffusion coefficient inside the nucleus (D = 5.6 ± 2.8 μm2/s) and in the cytoplasm (D = 8.6 ± 2.2 μm2/s). This is due to the different compositions inside these compartments, consisting of differing amounts of proteins, DNA and RNA.
Materials and Reagents
- Fission yeast cells expressing GFP in the nucleus (e.g. PD31 from Kalinina et al., 2013)
- Lectin (Sigma-Aldrich, catalog number: L2380 )
- MgCl2.6H2O
- CaCl2.2H2O
- KCl
- Na2SO4
- Pantothenic acid
- Nicotinic acid
- Inositol
- Biotin
- Boric acid
- MnSO4
- ZnSO4.7H2O
- FeCl2.6H2O
- Molybdic acid
- KI
- CuSO4.5H2O
- Citric acid
- Edinburgh minimal medium (EMM) (see Recipes)
Equipment
- Glass bottom dish (35 mm dish with a coverslip number = 1.5 and thickness 0.16 – 0.19) (MatTek, catalog number: P35G-1.5-14-C ) covered with Lectin.Spinning disk confocal microscope with a scanner-based FRAP system
- We use the Andor Revolution Spinning Disc System (Andor Technology), consisting of a Yokogawa CSU-X1 spinning disk scan head (Yokogawa Electric), which is connected to an Olympus IX81 inverted microscope (OLYMPUS). The microscope is equipped with a Prior ProScanIII xy scanning stage (Prior Scientific, Rockland MA, USA) and an Olympus UPlanSApo 100x/1.4 NA oil objective (OLYMPUS). Excitation for acquisition and bleaching is done using a Sapphire 488 nm solid-state laser (50 mW; Coherent). Laser power is controlled using the acousto-optic tunable filter in the Andor Revolution laser combiner (ALC, Andor Technology). The microscope is equipped with an iXon EM + DU-897 BV back illuminated EMCCD camera, cooled to -80 °C, pre-amp gain 2.4, EM gain 300 (Andor Technology). The resulting xy-pixel size in the images is 129 nm.
Note: Prior to the FRAP experiments, calibration has to be done following the FRAP calibration routine of Andor iQ software using the Andor FRAPPA calibration slide. Briefly, the software guides the user through a series of point-bleach steps during which one has to indicate the bleached point location in the image. This way the scanner bleach position is calibrated with the point position in the image.
- BL HC 525/30 (Semrock)
- Spectrometer
Software
- Andor iQ2 software (Andor Technology)
- Fiji (http://fiji.sc/Fiji)
- Matlab (MathWorks) software
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Delivani, P., Chacón, M. R., Schroth-Diez, B. and Tolić-Nørrelykke, I. M. (2013). Fluorescence Recovery After Photobleaching (FRAP) in the Fission Yeast Nucleus. Bio-protocol 3(20): e941. DOI: 10.21769/BioProtoc.941.
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分类
细胞生物学 > 细胞成像 > 荧光
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