For green Dendra2 add 486 to 553 nanometers, and for red Dendra2 add 580 to 740 nanometers.Īdjust the power and gain of both of the channels according to the intensity of the fluorophore and set the pinhole to fully open.
AGING PROTEIN TURNOVER SOFTWARE
When enough slides have been prepared, open the confocal microscope software and set the light path for excitation and emission. When the agarose has dried, carefully remove the top slide. Carefully add a few drops of agarose onto a clean glass slide and immediately place another slide on top of the drops to create a thin pad of agarose between the two glass pieces. While the agarose is cooling, cut the tip of a one milliliter pipette tip to facilitate aspiration of approximately 400 microliters of the melted agarose. On the day of the imaging experiment, melt general grade agarose at a 3%concentration in double distilled water. Begin by growing age-matched nematodes at 20 degrees Celsius to the desired age for the experiment. Take care not to provoke unwanted conversion of Dendra2 while scanning with the blue light laser, and to test and optimize the acquisition settings and conversion settings for each system and fusion protein.
To study, put in turnover within the proteostatis network as well as transport and trafficking. The method is adaptable to mammalian systems, as well as zebrafish. This in vivo and noninvasive technique requires only little microscopy set up to reliably convert Dendra2 into a bright and stable fluorophore.
This protocol allows for tracking and quantification of the degradation of a protein of interest in live and aging C.elegans.
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