I performed assays in microplates for the first time to determine enzymatic activity, but I made a mistake with the reading.
The recommended wavelenght for the assay is 550 nm. I used an ELISA reader (Robonik) with options for two filters. I programmed 578 nm in the primary filter and 550 nm in the secondary. I obtained interesting results, but I am not sure they are right because I have seen in some papers and my colleagues told me that the primary filter should be programmed with a lower wavelenght than the secondary filter. For example, in the case of my assay, primary: 492 nm and secondary 550 nm instead of 578 nm for the primary and 550 nm for the secondary.
It would wonderful to have some help with this question. Thank you very much!
Dear Mariana
which colorimetric substrate did you used to develope the elisa signal? Because the choose of the waveleghnt it is related to the substrate
https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-assays-analysis/elisa/elisa-reagents-buffers/enzyme-substrates-elisa.html
regarding the primary and secondary filter i never used robonik instrument so it is difficult give ti you a suggestion.
2 filter means--> 2 different results 1 for each waveleght?
because in this case also if you instrument provide to you 2 numbers, you need just one, the one that correspond to the absorbance of your substrate and it is important that this is that you acquire this waveleghnt.
good luck
Manuele
Thank you very much for the answers Manuele Martinelli and Achim Recktenwald
It is not a fluorescence assay. It is an enzymatic assay to determine B-1,3-glucanase activity.
With two filters I have only one result. The problem is that my equipment requires the use of two filters: primary and secondary. I know that I should adjust the secondary filter with the wavelength required for the assay (550 nm for reading reduced sugars), but I do not know what value to adjust in the primary filter. I am not sure if my results are correct because of this situation.
Why do you need two filters?
Normally, two filters are used, if a fluorescence is measured. The lower wavelength is the excitation and the higher wavelength the emission band that is measured. I have used fluorescence spectroscopy a lot in my previous lives to measure enzyme activities.
So, if your assay is using 2 filters, but is not a fluorescence-based assay, then I really would like to know what technique you are using.
Achim Recktenwald My assay protocol does not require two filters. It recommends to read at 550 nm (for chitinase, for example), but when I adjust the wavelenght in my equipment it requires primary and secondary values. I always put the recommended wavelength (550 nm) in the secondary, but I am obligated (by the reader) to adjust a value for the primary filter too.
I obtained interesting results with 578 nm in the primary and 550 nm in the secondary, but I am very insecure about them.
I had a quick look at the manual I found online, but it does not really explain the primary & secondary selection and what it does.
I would contact the vendor or manufacturer and ask them. They sgouild know their instrrument.
Thank you very much for your attention Achim Recktenwald
DEar Mariana
From the manual (attached) seems that, as i suspected your instrument is able to read 2 wavelenght in parallel (if you would like) therefore for some specific measurment you can select a primary ans a secondary filter on you slot. If i undertand well, you can also use a single filter setting the primary at wavelenght that you prefer eg 550nm and the secondary at zero.
However in some set-up cases you will obtain 2 results (eg. page 82) for each point but the number that you need to use is the one at the correct wavelenght, and in your case seems the value correspondig to the secondary.
however to give more suggegestion we need to se a printscreen of your insturment set up and your values.
good luck
Manuele
Thank you very much for the manual and the tips Manuele Martinelli
In photometric plate readers, two wavelengths are used. One is the absorbance maximum of the compound of interest (or close to it, if a filter for the correct wavelength is not available). The second should be set to the isosbestic point of that compound, i.e., the wavelength where absorbance is independent of concentration. Ideally, the reading at this wavelength should be constant for all samples, but because of imperfections in the plates (schlieren, scratches...) they are not. By measuring the ratio of absorbance at both wavelengths such artifacts can be corrected for. I have discussed photometry in chapter 5 of my "Biophysical chemistry of proteins" (doi:10.1007/978-1-4419-7251-4_5).
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