FRET based inhibition assay
Posted 11 March 2013 - 09:13 AM
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
Posted 11 March 2013 - 12:25 PM
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?
Posted 11 March 2013 - 03:38 PM
What filters are you using? Are they long pass or specific for the wavelength?
Posted 12 March 2013 - 01:28 AM
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!