something about ADP-Glucose Pyrophosphorylase Assay
 

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?

something about ADP-Glucose Pyrophosphorylase Assay
 

Don't you just hate when that happens?

Who here is a biochemistry expert?

Who can prognosticate how heavy the precipitation will be? (or is that
percipition?)

How mimsy are the borogoves?


robine wrote in message
m...
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?

something about ADP-Glucose Pyrophosphorylase Assay
 

Just guessing, I would say dirt. Some pertipitents will not form unless
there is something to start on, sometimes called a damon. Off the top of my
head I would think test tubes would be dirtier, we're talking micrograms
here, than a cuvette.

The borogoves have been gimbeling on the waybe too much.

"robine" wrote in message
m...
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?

something about ADP-Glucose Pyrophosphorylase Assay
 

can anyone give me some clues to figure it out?

The mome raths outgrabe.
Iris,
Central NY, Zone 5a, Sunset Zone 40
"If we see light at the end of the tunnel, It's the light of the oncoming
train."
Robert Lowell (1917-1977)

something about ADP-Glucose Pyrophosphorylase Assay
 

Now, that says it all!!!!

`Twas brillig, and the slithy toves
Did gyre and gimble in the wabe;
All mimsy were the borogoves,
And the mome raths outgrabe!

Just let Rinkytink argue over that!!!!


Iris Cohen wrote in message
...
can anyone give me some clues to figure it out?

The mome raths outgrabe.
Iris,
Central NY, Zone 5a, Sunset Zone 40
"If we see light at the end of the tunnel, It's the light of the oncoming
train."
Robert Lowell (1917-1977)

something about ADP-Glucose Pyrophosphorylase Assay
 

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

something about ADP-Glucose Pyrophosphorylase Assay
 

the glass test tubes are for one time use. so everytime i used a brand
new test tube. for the plastic cuvette, they are also new. so i do not
think it is the problem for the test tube or cuvette themselve.



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

something about ADP-Glucose Pyrophosphorylase Assay
 

the glass test tubes are for one time use. so everytime i used a brand
new test tube. for the plastic cuvette, they are also new. so i do not
think it is the problem for the test tube or cuvette themselve.



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