1.0 1233219362876 OMERO.editor PROTOCOL_ARCHIVE FRAP of Centromeres In order to measure the mobility of centromere proteins, bleach individual centromeres and follow recovery. This is tricky as the centromeres move quickly and the recovery time of some centromeric proteins can be quite slow (several minutes). Seed Cells Seed date: [[0]]. Seed 4 drops of HeLa into 2 ml of Penn-Strep-free media in 55 mm MatTech dishes Seed date 0 OPTIONAL DATE_TIME 1233568808174 1234568808174 1235568808174 SINGLE_STEP Transfect Next day, transfect each dish with [[1]] of GFP-construct:[[2]] using Oligofectamin protocol and reagents (Qiagen DNA 1 OPTIONAL NUMERIC 0.1 0.2 0.25 ug DNA construct 2 OPTIONAL TEXT SINGLE_STEP Imaging parameter-list Microscope: [[3]]. Used [[4]] with [[5]] binning. Took [[6]] z-planes at [[7]] spacin Microscope 3 OPTIONAL ENUMERATION 2nd-floor DV 2nd-floor DV 2nd-floor DV Mez DV pixel dimensions 4 OPTIONAL TEXT 256 x 256 binning 5 OPTIONAL TEXT 1 x z-planes 6 OPTIONAL NUMERIC 3 z-sections z-spacing 7 OPTIONAL NUMERIC 0.5 uM SINGLE_STEP Filters were [[8]], exposure of [[9]] with [[10]] ND filters filter 8 OPTIONAL ENUMERATION FITC DAPI FITC Red Far-Red exposure 9 OPTIONAL NUMERIC 0.1 0.1 seconds ND filter 10 OPTIONAL ENUMERATION 50 % 1 % 10 % 33 % 50 % 100% SINGLE_STEP FRAP parameters Take [[11]] before bleaching. Bleach with 50% laser-shot for [[12]] with [[13]] l pre-bleach images 11 OPTIONAL NUMERIC 3 images bleach duration 12 OPTIONAL NUMERIC 0.1 0.1 seconds laser 13 OPTIONAL TEXT 488 SINGLE_STEP Image recovery by taking an image every [[14]] for up to 10 minutes, or until centromere is lost from view. Meanwhile, maintain focus manually between exposures if necessary. Only do this if the middle of 3 z-sections is not the most in focus. time lapse 14 OPTIONAL NUMERIC seconds SINGLE_STEP Analysis Make a summed-intensity projection of the FRAP movies. Analyse the total signal in a circle (15 pixel diameter) centered on the centromere before bleach, after bleach and for every recovery image. Also take a background measurement. SINGLE_STEP