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FRET based inhibition assay


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#1 claire84

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Posted 11 March 2013 - 09:13 AM

Hi everybody,
I'm trying to set up a FRET-based inhibition assay to test some potential inhibitors against HIV-1 aspartyl protease. I monitor the increase of fluorescence intensity due to the cleavage of the peptidic substrate. If there is inhibition I should see a decrease of fluorescence. I'm using the fluoSTAR BMG plate reader.
I have problems because I'm obtaining noisy signals and and a low reproducibility between each replicate.
I'm using a plate mode set up and a top optic mode. There is also the possibility to set the gain.
Usually, you should set your gain on the basis of your positive control, but in this case fluorescence is not present in the positive control at the beginning of the measurement. In fact the fluorescence is generated during the enzymatic reaction. The first time I did the experiment I monitor the fluorescence of the positive control after 30 minutes and I used this number as the gain for the next experiments, but I'm not sure it's the correct way.
Can you help me? Do you have any experience with this kind of plate reader?

thanks a lot

#2 bob1

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Posted 11 March 2013 - 12:00 PM

I've used the fluostar - what do you need to know?

#3 claire84

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Posted 11 March 2013 - 12:25 PM

Hi,
I would like to know if there is something wrong in my protocol and how I can improve my measurements in term of signal noise and reproducibility. I was wondering if it's better to use the bottom optic instead of the top one and if the method I used to adjust the gain is correct. Can you suggest me any tips to improve my measurements?
Thanks :)

#4 bob1

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Posted 11 March 2013 - 03:38 PM

I assume that you are using black walled plates to eliminate the possibility of side-scatter? Make sure there aren't any bubbles on the surface of the wells, this will cause a lot of noise. For the gain I would use the well adjustment function under the "gain adjustment" tab in the read plate window. You should click on the well that will have the brightest fluorescence and use that to set the gain to 90% of the value given. I don't know how this would work for your system, but there are a series of videos on the BMG website that may give you more information.

What filters are you using? Are they long pass or specific for the wavelength?

#5 claire84

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Posted 12 March 2013 - 01:28 AM

Yes I'm using black plate (the bottom is black as well) and I paid attention to bubbles as well.
So for the gain, my method is not correct?
You are saying that is better to assume the positive control (protease+substrate without inhibitor) as the well will produce the highest fluorescence instead of to insert a number, right?
The filters are long pass.

Thanks I will have a look to the videos!




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