Frequently Asked Question's:
Q. What kind of test tubes should I use
with my Optocomp luminometer?
A: Polystyrene, polypropylene and
glass test tubes can all be used, however there are some important
considerations. Polystyrene test tubes can accumulate static
electricity, which can cause erratic high backgrounds when it
discharges. This effect can be minimized by carefully wiping
the outside of each test tube with a clean lint-free wipe (such
as a Kim-Wipe) lightly moistened with water prior to inserting
it into the instrument. This removes the static charge from the
exterior of the test tube. Glass tubes will not suffer from static
electricity, however glass tubes can phosphoresce after exposure
to fluorescent light or sunlight (any light containing ultraviolet
light). The phosphorescence can also cause elevated background.
If you are considering glass tubes, be sure to select tubes made
from borosilicate glass. Borosilicate glass usually has lower
levels of the contaminants that phosphoresce, however, some borosilicate
glass test tubes will phosphoresce. You can check for phosphorescence
using the method described in Tip 4, below. Polypropylene tubes
usually do not suffer from either static electricity nor phosphorescence.
Q. What is the minimum sample volume I can use in an Optocomp?
A: For the Optocomp I and II; anywhere from virtually no
sample up to about 1.5 ml (1,500 microliters) will work well.
Above about 1.5ml part of the sample will not be directly exposed
to the PMT, however this may not affect your results, as long
as all of your samples are the same volume and are reasonably
homogeneous. Sample volumes above 1.5ml are not an issue if you
are not attempting to quantify the analyte. For the Optocomp
P the range would be from virtually no sample up to about 1.1ml
Q. Are there any reagents that are not compatible with the
A: The fluid path in the Optocomp's injectors is constructed
entirely of Teflon and PEEK, two extremely inert materials. The
injector system is compatible with all of the popular reagent
systems and chemiluminescent and bioluminescent assay kits.
Q. What is an RLU?
A: RLU stands for "Relative Light Unit". This term
is used because luminometers typically do not yield a measurement
directly in units of photons. This is because some luminometers
measure the current output by the photomultiplier tube, which
cannot be translated into photons, while in the case of photon-counting
instruments, the effective bandwidth of the counters used to
accumulate the photon count is usually extended by dividing (pre-scaling)
the signal from the photomultiplier tube by some constant (usually
10) using a high-speed counter. Additionally, a calibration factor
is typically used to normalize the response of the instrument
to a calibrated light standard.
Q: My Optocomp I (or Optocomp P) does not have "Dual
Read" software. How can I best run Promega's Dual-Luciferase
Reporter assay (DLR assay) on my Optocomp I?
A: The Dual-Luciferase
Reporter assay is actually very flexible, and can be run with
a variety of luminometer set-ups. The DLR assay can be run using
an Optocomp I with or without injectors, or even on an Optocomp
P, which is not available with injectors. If you have two injectors,
you can have the instrument add both the Luciferase Assay Reagent
II (or LAR II, which initiates Firefly Luciferase luminescence)
and the Stop & Glo reagent (which quenches the Firefly Luciferase
luminescence and initiates Renilla Luciferase luminescence) to
your samples. If you have only a single injector, then you can
either use the injector to add one of the reagents and manually
(pipette) add the other, or you can change reagents used in the
injector in the middle of the assay. In either case (one or two
injectors) the injector(s) should both be set to deliver 100
microliters of the reagent. Or, you can use an Optocomp without
injectors, adding both reagents manually to your samples.
In any case, you should program two protocols
for the assay. One protocol will measure Firefly Luciferase in
your samples and the other will measure Renilla Luciferase. Simply
start the first protocol, and run all of your samples using the
LAR II reagent. Then start the second protocol and run all of
your samples again, this time using the Stop & Glo reagent.
The delay between addition of the LAR II reagent and the Stop
& Glo reagent will not affect your results. Of course, if
you are using injector(s) to add both reagents, then you need
to have the appropriate injector primed with the proper reagent
before starting each protocol.
Dual-Luciferase and Stop & Glo are trademarks
of Promega Corporation
Q. How can I determine how light emission
from my samples changes over time?
A: With the Optocomp I and the Optocomp
II, you can use the Kinetic protocol. You can program a Kinetic
protocol to divide each measurement into up to 100 time slices,
or intervals, with each interval programmable from 20 milliseconds
up to 650 seconds. After each sample is read, the Optocomp will
print the total RLU measured as well as the RLU for the peak
interval. You can then choose to plot a histogram of the light
emission, and/or print the data for all of the intervals.
Q: What kind of standard can be used to calibrate my Optocomp
A: A standard is available from MGM Instruments for this
purpose. The standard contains a very small amount (less than
10 microcuries) of Tritium. Tritium has a half-life of about
12 years, so the activity of the standard must be corrected for
decay of the Tritium. A decay chart is provided with each standard
for this purpose.
Q: What kind of standard can be used to
calibrate my Isocomp gamma counter?
A: A standard is available from MGM
Instruments for this purpose. The standard contains a very small
amount (less than a tenth of a microcurie) of I129, which is
used to simulate the I125 typically used in many radioisotope-labeled
assays. The energy levels of the x-ray and gamma radiation of
the two isotopes are very similar, making use of I129 practical.
I125's half-life (60 days) makes its use as a standard impossible,
while I129's half-life of 15.7 million years makes it a stable
source suitable for use as a calibrator.
It is extremely important that the Isocomp
is properly calibrated before running an assay. The Isocomp accepts
a narrow range of radioactivity based on its energy, this is
done to exclude background radiation from your measurements.
As a result, if the instrument is not properly calibrated, it
is quite possible that little or none of the actual signal might
be measured, causing serious error in the assay.
Optocomp is a registered trademark
of MGM Instruments, Inc.
1. Tip: To insure accurate results, you should calibrate
your Isocomp every day, before running assays!
2. Tip: To insure reliable performance from your Optocomp's
injectors, be sure to flush them out thoroughly immediately after
you are done using the instrument. Many reagents used with luminometers
will clog the fluid path if left in the injector. You can flush
your injectors out with DI water. If they have become clogged
try flushing them with warm DI water or a mixture of DI water
3. Tip: If you suspect your Optocomp I or Optocomp P has
background problems, try making a measurement without a sample
tube in the instrument. This will allow you to determine exactly
what the instrument's background is. This is important, because
we find that usually the problem is with the samples or the test
tubes, and not the instrument.
For the Optocomp I, first, start a protocol that does not have
injectors programmed in it (the count time should be set to one
second). Next, notice that there are two round black plastic
devices in the sides of the recess the lid closes into, to either
side of the sample position. One of these is an infrared emitter
and the other is a detector, these are used to detect the presence
or absence of the sample test tube in the measurement position.
With the lid completely open, place your finger over one (it
doesn't matter which) of these little round black plastic rimmed
holes. Now, holding your finger there, close the lid part way,
then remove your finger and finish closing the lid. The Optocomp
I will now be fooled into believing a sample tube is in the measurement
position, and will proceed to acquire it. To make repeated measurements
simply hold the lid closed.
To check the background in an Optocomp P portable luminometer
simply close the turret without placing a sample tube into the
turret. The Optocomp P does not have injector capability, and
so does not require a means to detect whether a sample tube is
Unfortunately, instrument background cannot be easily checked
in the Optocomp II.
4. Tip: To determine if your test tubes are phosphorescing,
run a raw data protocol programmed for a one second count time
and no injectors. After determining the instrument background
using the method described in the previous tip, hold each of
the test tubes you wish to check, empty, relatively close (two
or three feet away is fine) to a fluorescent light for several
seconds, then immediately and quickly place each tube into the
instrument and close the lid (in an Optocomp I), or rotate the
turret closed (in an Optocomp P). Hold the lid down and allow
the instrument to measure the test tube repeatedly across a period
of twenty or thirty seconds. Any phosphorescence will be seen
as an elevated background reading that will gradually decline
over the period of the measurement until normal instrument background
It is difficult to measure phosphorescence of test tubes in an
Optocomp II, because of the time it takes to place the tube(s)
into the cassette(s) close the lid and initiate the measurement.
Fortunately, because you typically load some number of tubes
into an Optocomp II and then start the protocol, any phosphorescence
will usually have decayed before the tubes are read. If you suspect
your measurements are being affected by phosphorescence of the
test tubes, then simply load your samples into the instrument,
close the lid, and wait a minute or so before starting the protocol.