claire84

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

Hi everybody!!
I'm doing some enzymatic kinetic studies and I need your help!!I'm using a FRET based assay to evaluate the strenght of some potential inhibitors against HIV-1 protease.
I wanted to measure the Km and the Vmax of HIV-1 protease for my peptidic substrate http://www.sigmaaldr...atalog/product/sigma/h0790?lang=it&region=IT and I performed different enzymatic reaction at 8 substrate concentrations. I used graphpad to obtain Km and Vmax, but the Vmax is expressed as RFU/sec and I know I should convert it as mol/min/mg of protein. How can I do this?
I think I should have a calibration curve of my product but I'm not sure...

I tried to perform a calibration curve using the anthranilic acid, but at the end the concentration I obtained was negative....Where is the mistake????

thanks a lot

Claire

Hello everybody,
I'm doing some enzymatic kinetics using the HIV-1 aspartyl protease. In particular I'd like to determine the Km of the enzyme for a particular substrate (http://www.sigmaaldr...ng=en&region=GB) in order to decide which substrate concentration to use for my inhibition assays.
I'm using a MES buffer (25mM) with NaCl (200mM), 5% Glycerol, 5% DMSO, 0.0002% triton X-100 and 1mM DTT.

I should measure the increase of fluorescence due to the activity of the protease.

During the measurement I encoutered some problems regarding the signal of the blank (buffer+substrate). It's very high and noisy.
is it possible some of the buffer components show fluorescence at the same wavelenght of the substrate? (ex: 320nm, em:420nm)
Or maybe is the problem related to the stability of the signal?The wells where is the blank are the first that are measured....

Any suggestions??

Thanks a lot!

Hallo everybody,
I'm going to start the set up an enzymatic inhibition assay, but I'm not an expert and I'm having some problems to understand some key points of these assays.
In particular, I understood I need to run the reaction under initial velocity condition, but how can I measure it?
I know it's the initial linear portion of the enzyme reaction when less than 10% of the substrate has been deplcted, but esperimentally how can I decide it?

I'll really appreciate your help and suggestions
thanks a lot!

Hi guys,
maybe it's a silly question, but I'm not a cellular biologist and I need some help.
I isolated PBMC from the whole blood and then I seeded them in a 96 well plate. After incubation of 24h I would like to monitor the presence of a certain protein on their surface using a labelled antibody.

Do I need to treat them as adherent cells using trypsin before adding the mix with the antibody or can I resuspend them pipetting?
What do yo think?

thanks a lot!