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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...

COLORIMETRIC PORPHOBILINOGEN ASSAY (EHRLICH) Modified Ehrlich Reagent Dissolve 1.0 gm p-dimethyl aminobenzaldehyde in 30mL glacial acetic acid. Add 8.0 mL 70% perchloric acid. Bring to 50 mL with glacial acetic acid. Store in the dark at 4˚C for about 2...

BLOOD UROD   Sample preparation Draw two green-topped collection tubes of blood.  These tubes have heparin as   anticoagulant. Spin down the RBCs (red blood cells) at 2600rpm for 5min at 4˚C. Discard the plasma and buffy coat. Wash RBCs twice by fillin...

BLOOD HEMOGLOBIN ASSAY (DRABKINS)   Drabkins Reagent Dissolve 1.0g NaHCO3, 0.05g KCN and 0.20g K3Fe(CN)6 in some water. Bring to 1.0L final volume. Store refrigerated. Hemoglobin Assay (formation of cyanomethemoglobin) Pipet 5.0mL Drabkins reagent...

d-AMINOLEVULINIC ACID SYNTHASE (ALAS) Mouse liver homogenate Weigh out ~100mg liver. Add four volumes (400µL) of ice-cold 50mM potassium phosphate (KPi) pH7.4. Homogenize with ten up-and-down strokes in a 2-mL glass-Teflon (Potter- Elvehjem) tissue homog...

AMINOLEVULINIC ACID DEHYRATASE (ALAD)   Mouse liver homogenate Weigh out ~100mg liver. Add 400 µL 1mM DTT (dithiothreitol) in 50mM KPi (potassium phosphate) pH 6.8. Homogenize with ten up-and-down strokes in a 2-mL glass-Teflon (Potter- Elvehjem) tissue...

Normal operation of the StedyCon microscope through a simple confocal scan.  Separate pages will cover other types of scans, e.g., Z-Stacks, Tiled scans, STED. These documents are to reinforce, not replace, hands on training on the microscope.  All users MUST...

Basic instructions for the EVOS M5000 in Crocker 32. The EVOS M5000 The EVOS M5000 is capable of imaging slides in brightfield, phase contrast, and/or epifluorescence.  It can also do Z-stacks and projection as well as timed experiments.  Unlike the EVOS Aut...

PCR Products Quantitate using a gel (nanodrop isn’t reliable with PCR products) For small templates use 50ng PCR product (please clean it up first as left over primers can mess up sequencing) and 4 picomoles of primer (4uL of a 1uM aliquot) For larger tem...