Rlu luminescence dating
To check the background in an Optocomp P portable luminometer simply close the turret without placing a sample tube into the turret. Polystyrene test tubes can accumulate static electricity, which can cause erratic high backgrounds when it discharges.
The Isocomp accepts a narrow range of radioactivity based on its energy, this is done to exclude background radiation from your measurements. It is extremely important that the Isocomp is properly calibrated before running an assay. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters.
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. This is important, because we find that usually the problem is with the samples or the test tubes, and not the instrument. Or, you can use an Optocomp without injectors, adding both reagents manually to your samples. 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. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.
The standard contains a very small amount less than a tenth of a microcurie of I, which is used to simulate the I typically used in many radioisotope-labeled assays. The injector system is compatible with all of the popular reagent systems and chemiluminescent and bioluminescent assay kits. The phosphorescence can also cause elevated background. The Optocomp P does not have injector capability, and so does not require a means to detect whether a sample tube is present.
To determine if your test tubes are phosphorescing, run a raw data protocol programmed for a one second count time and no injectors. Additionally, a calibration factor is typically used to normalize the response of the instrument to a calibrated light standard. Polypropylene tubes usually do not suffer from either static electricity nor phosphorescence. The simple and cheap assay can easily be adapted to a variety of experimental situations. The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research.
To normalize the values, you divide each firefly luciferase value by the value for the renilla in the same well. 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.
To analyze these numbers, you usually use a control, to which your samples are then relative to. Borosilicate glass usually has lower levels of the contaminants that phosphoresce, however, some borosilicate glass test tubes will phosphoresce.
This will allow you to determine exactly what the instrument's background is. They are influenced but the kit you use for the measurement, the machine you use so always use the same machine and so on.
Any phosphorescence will be seen as an elevated background reading that will gradually decline over the period of the measurement until normal instrument background is reached. This term is used because luminometers typically do not yield a measurement directly in units of photons. If you suspect your Optocomp I or Optocomp P has background problems, try making a measurement without a sample tube in the instrument.
Polystyrene, polypropylene and glass test tubes can all be used, however there are some important considerations. In the example the relative expression of sample B would be half the one of the control. 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. In any case, you should program two protocols for the assay.
To insure reliable performance from your Optocomp's injectors, be sure to flush them out thoroughly immediately after you are done using the instrument. The Optocomp I will now be fooled into believing a sample tube is in the measurement position, and will proceed to acquire it. They are the result of the luciferase reaction and proportional to the expression of the luciferase gene, meaning the higher the numbers are, the higher the expression is.
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