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Location: EEJMRB Room 5122 Hardware: Leica DMi8 motorized microscope with adaptive focus tracking ASI motorized stage with Piezo Z W-1 large format spinning disk confocal with beam homogenizer Andor 1024 x 1024 EMCCD camera Visitron 2D FRAP unit 405,4...

Location: HSC Room 56 Main features of System: Four Photo Multipliers Tube (PMT) detector unit enables simultaneous 12 bit (4096 intensity levels) acquisition of up to 4 fluorescent channels. Fifth transmitted light detector available for simultaneous b...

Location: HCI Room 1430 Main features of System: Widefield light pathway employs a high sensitivity Andor Zyla CMOS camera Sola automated LED lightsource for both widefield fluorescence and transmitted illumination pathways Fully automated Nikon Ti-E inv...

Location: HSC Room 48B / 48D Main features of Prairie Ultima 2/Multi-Photon Confocal System: Six detectors, four reflected mode Photo Multipliers Tube (PMT) detectors and two transmitted mode PMTs for second harmonic and fluorescence detection. High NA wa...

Location: CSC Room 30 Main features of StedyCon: STEDYCON from Abberior is a new class of nanoscope with multicolor confocal and STED (Stimulated emission depletion) capabilities. The lateral STED resolution could reach 30nm depending on dyes and objective...

A scan profile describes how the scan will be done (objectives, channels, exposures, etc).  The Advanced Profile Wizard is one way to set up or modify a scan profile. Brightfield vs Fluorescent Profiles Brightfield profiles are simpler than Fluorescent profi...

Work Auth System Overview Prerequisite to gaining access to the cores resource system or being able to request services from any cores managed facility you will be required to fill out a "Work Authorization" form. This form is a digital authorization and data ...

UROPORPHYRINOGEN DEAMINASE (UROD) ASSAY   Reaction A: Uroporphyrinogen (substrate) synthesis   Add 10 µL 0.5 µg/µL rPBGD (recombinant porphobilinogen deaminase) stock to   75µL 10mM DTT (dithiothreitol) in 0.1M Tris pH 7.65.  (This is for the           s...

ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (UPLC) QUANTITATION OF PORPHYRINS Inject 10µL of supernatant solution of porphyrins in 1.5M HCl into a Waters Acquity UPLC system which includes a binary solvent manager, sample manager, fluorescence detector, column ...

UROPORPHYRINOGEN III SYNTHASE (U3S) Cytosolic U3S Assay Mix 100 µL 20mM Tris pH 8.2, 260 µL 20mM KPi (potassium phosphate) pH 8.2 and 20 µL 0.5mg/mL rPBGD (recombinant porphobilinogen      deaminase). Mix well and incubate for at least 2 min at 37˚C. Add...

Sonicate the washed pellet of cultured bacterial cells in two volumes of 10mM KPi pH 5.5. Determine protein content and adjust to about 20mg/mL with the same buffer to prepare the homogenate.  Deoxygenate all samples and reagents. Mix about 50µL homogenate ...

HEME QUANTITATIONS FROM PYRIDINE HEMOCHROMOGEN SPECTRA   Hemochromogen formation   Prepare a pyridine reagent mix by adding 3mL 1M NaOH and 6mL pyridine (use glass pipet) to 19mL H2O in a glass container (28mL total volume). Put 1000µL of the pyridine r...

PROTOPORPHYRINOGEN OXIDASE Cultured cell homogenate Resuspend the pellet of PBS-washed cells in about three volumes of 50mM potassium phosphate pH 7.4 (KPi). Sonicate (homogenize) while in an ice bath at the lowest practicable power setting for 3 cycles x...

CHEMICAL REDUCTION OF PORPHYRINS USING PALLADIUM CARBON Put 5.0mg Pd/C (palladium carbon) and 2.0mg porphyrin in a 13 x 100mm borosilicate test tube.  Put the tube on a suitable tube rack. Wet the contents with 0.2mL water to avoid potential explosion when...

PORPHOBILINOGEN DEAMINASE (PBGD)   PBGD Assay Add 10µL homogenized sample to 75µL in 0.1M Tris pH 7.65. Add 15 µL 2.2mM PBG (porphobilinogen) in 0.1M Tris pH7.65. Incubate in a 37˚C water bath for 30 min. Stop the reaction with 100µL 3M HCl. Expose th...

HEMIN, PROTOPORPHYRIN IX AND ZINC PROTOPORPHYRIN IX EHTYL ACETATE / ACETIC ACID EXTRACTION AND UPLC   Solvent Extraction Prepare extraction solvent (EA) by mixing four volumes of ethyl acetate to one     volume of glacial acetic acid. Dilute sample homo...

FERROCHELATASE (FECH) Sample preparation Suspend ~50-µL mammalian cell pellet in 400µL TGD buffer (Tris buffered glycerol with dithiothreitol (DTT), made by dissolving 2mL or 2.52 g glycerol and 1.5mg DTT in 8.0 mL 20mM Tris pH 8.0) Sonicate (homogenize) ...

COPROPORPHYRINOGEN III OXIDASE (COPOX) ASSAY Reaction A: Uroporphyrinogen III synthesis Add 8 µL 0.5 µg/µL rPBGD (recombinant porphobilinogen deaminase) stock and 2µL 1µg/µL rU3S (recombinant uroporphyrinogen III synthase) to 70µL    10mM dithiothreitol in...