In vitro proline dehydrogenase microtiter assay

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In vitro proline dehydrogenase microtiter assay

In vitro proline dehydrogenase microtiter assay

STOCK SOLUTIONS:
4X reaction buffer.
0.76 g Tris HCl
4.20 g Tris base
40 ml ethylene glycol
1 ml Tween-20
q.s. 100 ml

INT (p-iodo-nitrotetrazolium violet) (3.2 mg ml -1).
Add 160 mg INT to 50 ml dH20.
Heat to 80oC to dissolve.

L-proline stocks.
Stock (M) 1 M L-proline (ul) dH20 (ul) Reaction (mM)
1.00 500 - 100
0.80 450 50 80
0.60 400 100 60
0.40 350 150 40
0.20 300 200 20
0.10 250 250 10
0.08 200 300 8
0.00 - 500 0

REACTION:

1. Prepare stock mixture: 2.5 ml 4X reaction buffer
1.6 ml INT stock
4.9 ml dH2O
2. From this stock add 85 ul to each well of a microtitre plate.
3. Add 10 ul of the appropriate proline stock dilution (see plate below).
4. Start reactions in triplicate (1-3, 4-6, etc.) by adding 5 ul of PutA (diluted to 0.1 ug ul -1) to the appropriate wells.
5. Incubate 5-10 min (or until red color change) at 37oC.
6. Stop reactions with 100 l of 2N HCl.
7. Read at OD490 and correct each reaction with the appropriate control.
8. Calculate activity as shown below:

Specific activity (nmol INTR min -1 mg -1 ) =
OD490 0.1 ml
-------------------------------------------------
6.64 X 10-3 ml -1 (5 min) (5 X 10-4 mg)

Link: http://www.sci.sdsu.edu/~smaloy/Research/pdf%20files/Pro_DH_in_vitro_-_microtiter.do.doc