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Testing

Detection of Membrane Localization of Halorhodopsin by Immunohistochemistry

Sep. 15

1ml cyanobacteria liquid for each sample was prepared, including four experiment groups--Pdark-HR(green), Pdark-HR(red), PcpcG2-HR(green) and PcpcG2-HR(red), which had been cultured to OD730=0.6 and induced for 12h, and controls—wildtype(green) and wildtype(red), which were not been induced. Anti-His mAb was applied with PBS in proportion of 1:100 as the first antibody. After 2 hours’ incubating(at 37℃) and washing steps, goat anti-mouse IgG Alexa Fluor 488(green fluorescence) and goat anti- IgG Alexa Fluor 594(red fluorescence) were applied respectively with PBS in proportion of 1:1000 as the second antibodies. After 2 hours’ incubating(at 37℃) washing steps, the cyanobacteria pellets were resuspended with 50μl PBS. Six slides were prepared. While observing under a confocal microscope, we found out that the cyanobacteria has spontaneous red fluorescence. Several positive results can be seen but the signal is not strong enough. So we decided to optimize the protocol and redo it.

Sep.15 evening to Sep. 16

1ml cyanobacteria liquid for each sample was prepared, including four experiment groups--Pdark-HR(1:100), Pdark-HR(1:50), PcpcG2-HR(1:100) and PcpcG2-HR(1:50), which had been cultured to OD730=0.6 and induced for 16h, and controls—wildtype(1:100) and wildtype(1:50), which had been induced for 16h as well. The first antibody anti-His mAb was applied with PBS in proportion of 1:100 or 1:50 as mentioned. The samples were incubated at 4℃ overnight. The second antibody goat anti-mouse IgG Alexa Fluor 488(green fluorescence) was applied with PBS in proportion of 1:300 and the samples were incubated at 37℃ for 2 hours. After washing the samples, the cyanobacteria pellets were resuspended with 20μl PBS. Six slides were prepared. The results were better than the former one. Concerning of time, we only got the micrographs of the samples containing the first antibody with concentration of 1:50.

图片

In the experiment groups (Pdark-HR and PcpcG2-HR), many loops of green fluorescence can be seen under the confocal microscope while the control groups (wildtype) cannot, which indicates that the halorhodopsin expressed successfully on the cell membrane of the modified cyanobacteria instead of any other position in the cells.