Core tips:
Key words: fluorescent protein labeling
Key words: GFP subcellular localization of LSCM
1、 Principle
Using green fluorescent protein (GFP) to trace intracellular protein. At present, GFP fusion protein technology is a popular method to study the location of living cells. And the molecular weight of GFP is 27 KD. After laser irradiation by laser scanning confocal microscope, it can produce a green fluorescence, which can accurately locate the protein.
Laser scanning confocal microscope (LSCM, hereinafter referred to as confocal microscope) because of its unique design principle, effectively eliminates the non focal plane information, improves the resolution and contrast, and makes the image more accurate and clear, so it is extremely suitable for the positioning of proteins, nucleic acids and other living cells and dynamic research in vivo.
2、 Main steps
- Construction of eukaryotic expression vector
① Primer design
Using primer design software, the primers of the target gene were designed according to the cleavage site of pEGFP-N1
② Carrier construction
The PCR product was digested and inserted into pgfp-n1 to obtain the eukaryotic expression vector of the fusion protein of the target gene and EGFP.
- Transfection of eukaryotic cells
When the cells grew to the logarithmic growth stage, they were inoculated into a special glass plate (35mm Petri dish, 10 mm microwell) for confocal microscope and cultured overnight. When the cell adhesion rate reaches 30% ~ 50%, the expression vector plasmid 2ug and Lipofectamine 2000 2ml are respectively dissolved in 100 ml DMEM medium without antibiotics and serum. After fully mixing, the two solutions are placed at room temperature for 15 minutes, and then the two solutions are fully mixed, and placed at room temperature for 30 minutes. At the same time, DMEM without serum and antibiotic was used to wash the culture cells to be transfected for 2-3 times. 800 ml DMEM without antibiotic and serum was added to the DNA liposome mixture, and then the mixed DMEM was added to the culture cells. The dish was incubated in a 37 ℃ incubator for 6-8 hours, then the double culture medium was sucked out, and 2-3 ml DMEM complete medium containing antibiotics and 10% FCS was added, and the culture continued for 24-72 hours.
- Laser scanning confocal microscopy
The cells were observed by confocal microscope at 24, 36, 48 and 72 hours respectively. The laser scanning confocal microscope was used. The excitation wavelength was 488 nm. The image was taken.
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