GST activity - (May/18/2008 )

hi friends,

Currently I working on herb-drug interaction research.

I face dificulty to conduct GST assay and to calculate GST activity.

I conduct my assay using microtiterplate.

The final volume is 250 uL. The final concentration of CDNB and GSH is 1mM.

The final cytosolic fraction used is 2 mg/ml ( I used 2 mg/ml because previous master student has done the optimization of the assay condition, and he found out that the optimum protein concentration is 2 mg/ml

All of above were disolved in 100 mM pH 6.5.

The equation I used is

Activity (umoles/min/uL) = Relative absorbance/min X Final assay volume x fold dilution of GST stock
----------------------------------------------------------------------------------------------------------
9.6 umole/mL X volume of enzyme

Relative absorbance/min= 0.38/min
Final assay volume = 250 uL
fold dilution of GST stock= 12 times dilute ( I made a stock of cytosolic fraction 10 mg/ml, final concentartion in the assay will be 2 mg/ml, 12 times dilute)
volume of enzyme = 60 uL

I calculate like this

GST activity= 0.38/min X 250 uL X 1/12
-----------------------------------------------
9.6 umole/ml x 60 uL

= 0.38/min X 250 uL X 1/12
---------------------------------
9.6 umole/1000 uL x 60 uL

= 0.38/min X 250 uL X 1/12
-------------------------------------------
0.0096 umole/uL x 60 uL

= 7.917uL min-1
------------------------------
0.576 umole
= 13.74 uL min-1
-------------------------
umole
=13.74 uL min-1 umole-1
= 13.74 uL/min/umole

Why the final unit is not umoles/min/uL ?

Are my calculations wrong?

Are protein optimization important before conducting the assay?

Because I have read an article that the cytosolic fraction for pig liver need to be diluted 1000 times. It's like every time they want to use pig liver cytosolic fraction, thye must dilute 1000 X regardless of the protein concentration

Is it true that absorbance reading must not exceed 0.8? if exceeded , I need to dilute more the cytosolic fraction?

For GST specific activity the unit is umole/min/mg.

GST specific activity= GST activity (umole/min/ml)
-------------------------------------------
concentration of stock of cytosolic fraction used

concentration of stock of cytosolic fraction used= 12 mg/ml

so

GST specific activity= GST activity (umole/min/ml)
-------------------------------------------
12 mg/ml

Is what i am doing correct?

Thank You

-kent19-

QUOTE (kent19 @ May 18 2008, 08:08 AM)
hi friends,

Currently I working on herb-drug interaction research.

I face dificulty to conduct GST assay and to calculate GST activity.

I conduct my assay using microtiterplate.

The final volume is 250 uL. The final concentration of CDNB and GSH is 1mM.

The final cytosolic fraction used is 2 mg/ml ( I used 2 mg/ml because previous master student has done the optimization of the assay condition, and he found out that the optimum protein concentration is 2 mg/ml

All of above were disolved in 100 mM pH 6.5.

The equation I used is

Activity (umoles/min/uL) = Relative absorbance/min X Final assay volume x fold dilution of GST stock
----------------------------------------------------------------------------------------------------------
9.6 umole/mL X volume of enzyme

Relative absorbance/min= 0.38/min
Final assay volume = 250 uL
fold dilution of GST stock= 12 times dilute ( I made a stock of cytosolic fraction 10 mg/ml, final concentartion in the assay will be 2 mg/ml, 12 times dilute)
volume of enzyme = 60 uL

I calculate like this

GST activity= 0.38/min X 250 uL X 1/12
-----------------------------------------------
9.6 umole/ml x 60 uL

= 0.38/min X 250 uL X 1/12
---------------------------------
9.6 umole/1000 uL x 60 uL

= 0.38/min X 250 uL X 1/12
-------------------------------------------
0.0096 umole/uL x 60 uL

= 7.917uL min-1
------------------------------
0.576 umole
= 13.74 uL min-1
-------------------------
umole
=13.74 uL min-1 umole-1
= 13.74 uL/min/umole

Why the final unit is not umoles/min/uL ?

Are my calculations wrong?

Are protein optimization important before conducting the assay?

Because I have read an article that the cytosolic fraction for pig liver need to be diluted 1000 times. It's like every time they want to use pig liver cytosolic fraction, thye must dilute 1000 X regardless of the protein concentration

Is it true that absorbance reading must not exceed 0.8? if exceeded , I need to dilute more the cytosolic fraction?

For GST specific activity the unit is umole/min/mg.

GST specific activity= GST activity (umole/min/ml)
-------------------------------------------
concentration of stock of cytosolic fraction used

concentration of stock of cytosolic fraction used= 12 mg/ml

so

GST specific activity= GST activity (umole/min/ml)
-------------------------------------------
12 mg/ml

Is what i am doing correct?

Thank You