PAPS (phosphoadenosine phosphosulfate) preparation
This protocol is used to prepare PAPS, a sulfate donor used in for example assays measuring the activity of sulfotransferases.
The enzymes needed to convert ATP and sulfate into PAPS are prepared from Yeast.
Yeast (Can be bought from example SIGMA or grown in the lab)
Radiolabelled sulfate (35SO4)
1 M Tris-HCl, pH 8
Yeast extract preparation
Dissolve 400 mg yeast in roughly 2 ml of 10 mM Tris-Hcl, pH 8
Add 5 ml of glass-beads
Vortex and hand shake for a total of 10 minutes, make sure to keep homogenate cold.
Recover material by letting beads sediment and adding 6 x 1 ml of 10 mM Tris-Hcl, pH 8.
Centriguge recovered homogenate at 20 000 x g for 10 minutes at 4 degrees
Dialyse three times against 1 liter of 10 mM Tris-Hcl, pH 8
Determine protein content
Aliquot and flash freeze in liquid nitrogen and stora at -80 degrees
Generating PAPS35 from ATP, sulfate and yeast extract
Pipette in following order:
200 microliter buffer 5X (250mM Tris-HCl pH 8.0 , 80mM ATP)
Make to 8 mM MgCl2 from stock or powder
Dialysed yeast extract to final 2 mg/ml
Add 1mCi 35SO4
Make to final 1ml with water
Incubate for 2 hours at 37 degrees
Add 5 ml water. Boil for 2 min (no longer: risk of hydrolysis of PAPS).
Centrifuge at 20 000 x g for 10 minutes as above
Separate radiolabelled PAPS from unincorporated sulfate and ATP on a DE-52 column.
You can expect a 30% incorporation of radiolabelled sulfate with this protocol when using dried yeast. Freshly grown yeast culture might improve this number considerably.
Modified by Dr. Marco Maccarana
Delfert D. M. et al, (1985) Anal. Biochem. 148, 303-310 (This cited paper use extract from chondrocytes and not from yeast)
Added by --Yourscic 11:36, 21 February 2007 (CST)