robine wrote:
I got something very unexplainable during the ADP-Glucose
Pyrophosphorylase Assay.
The samples which i analysed were collected from the cotton (1-2
flowers open) in greenhouse, including stem, hypocotyl and taproot.
All those samples were sliced to small disks and storage in -80 degree
freezer.

The extraction solution which includes 50mM HEPES,pH7.4,5mM MgCl2, 2mM
DTT (DL-Dithiothreitol), 1mL EDTA and 0.1%Triton-X is 1mL for each
sample.

The final Assay Solution which includes 50mM HEPES,pH8.0,10mM MgCl2,
2mM DTT (DL-Dithiothreitol), 5mM ADP-glucose ,0.4mM NAD ,1.8mM PPi
(Tetrasodium pyrophosphate), 2 units phosphoglucomutase , 2 units
glucose-6-phosphate dehydrogenase and 0.05mL extraction solution.

Final 0.9mL assay solution were placed in a Acryl-cuvette (Sarstedt)
before initiated by 0.1mL 18mM PPi.As soon as i mixed the PPi with the
solution together, sometimes there were percipition in the cuvette ,
heavily or little, sometimes there were no percipition in the cuvette
at all. Everything is the same but the extracted sample. I cannot
explain why this happened. If i mixed the solution in a glass test
tube, there always had percipiton in the test tube!

can anyone give me some clues to figure it out?



do you wash your glass test tubes in a dish washer? our old one didn't
rinse well. so glass often had a bit of a detergent residue. your
plastic cuvettes would be more disposable. have a look if you get
percipitation on a new plastic cuvette.
are the tubes or PPi chilled? i think some surfactants percipitate when
cool - SDS will very quickly in the fridge don't know about triton.
is it a large flaky percipitate (probably surfacant), white snotty
(protein/sugar) or clear (could be DNA)?

http://www.pubmedcentral.nih.gov/art...i?artid=102300
these people used more buffering 100mM HEPES?

craig