Set to probe I
before first use or make sure plugged into same port.
Open program
NEIHS chart (recorder) for windows.
Settings:
Channel 2
Hardware=DT280
Address=2EC
Printer=epr8/hplaser4050p5
at 300dpi and landscape across the hall in office
Set time to 4-5
minutes to allow time for baseline and sample input.
Computer
Setup:
Click
experiment, data and set to chart mode.
Set on
continuous reading and data collect.
Click
experiment, chart recorder. Set oxygraph to amp zero and set needle to zero
with coin then hit measure 0% in program to set. Set oxygraph to air and adjust
with coin to 100% then click 100% to set.
Measure
samples as follows:
Make sure
oxygraph is set to air before adding sample.
Add COX-2 and
Hematin (and inhibitor if necessary) to a tube containing 2ml of 1X buffer on
ice.
Remove samples
from ice and add to reservoir (2.0ml) then click data collect.
Wait 1 minute to
allow for baseline measurement of sample, then add AA to 100uM (f.c.) and allow
it to finish the timed run.
Save as .exp
then open as text file in notebook and save again. Repeat with others.
COX-2
enzymatic assay: COX-2
activity was determined by measuring Oxygen consumption at 37’C in an oxygraph
chamber with an oxygen electrode. The reaction buffer consisted of 100mM Tris,
pH 8.0 with 500uM phenol. For estimating the Km for arachidonic acid, the
solubilized COX-2 (100-500ug enzyme) were reconstituted for 1-min with 10uM
hematin on ice. Subsequently, they were incubated for 1-min at 37’C in reaction
buffer before being challenged with final concentrations of 100uM arachidonic
acid.
240uM O2 at 37’C
is saturated. Multiply by two per AA molecule and 2 for oxygen atoms if
desired. 10X buffer=1M Tris pH8.0 with 5mM Phenol. F.C.= 100uM AA; 10uM
Hematin; 100-500ug Cox-2; 100mM Tris pH8.0; 500uM Phenol.
AA oil is 3.29M;
use 30.5ul/ml. AA in etoh is 300uM; use 333ul/ml.
See Jean Corbett
or Charles Detwiller in Dr. Ron Mason’s laboratory for help.