So I am doing a HTS screening to select top drugs for investigating the nucleo-cytoplasmic shuttling of my target protein. The HTS assay involved drug treatment of the cells expressing GFP-tagged fusion proteins and from the screen, I chose the top targets which had the highest nuclear: cytoplasm ratio. Then I validated them in vitro through cellular fractionation and Western blotting. But its turns out that all of them are negative - none of the drugs showed an increased nuclear presence of my target proteins? How can I critically argue this? I will be glad if any one can suggest any improvements?
Hi - not too familiar with your 'shuttling assay' but I can guess that something is going on when you are fractionating your nuclear and cytoplasmic components. These fractionating buffers/kits do work for proteins but I've not seen it work for small molecules. Once you add your buffer in the first step (i.e. lyse cell membrane but not the nuclear membrane) you are creating a large osmotic gradient, so what's to stop your compound flying out of the nucleus again? The 'shuttling assay' does suggest a two way street... I think you need to rethink your validation assay, where you are keeping the cells intact. Do the molecules/HTS hits fluoresce i.e. UV spectrum? perhaps you could find a suitable excitation wavelength and look at it via live imaging microscopy? Apologies, haven't done this myself, perhaps someone else has a better idea....
Allow me to explain my strategy. I used a drug screen from NIH that consists of 118 drugs. For my assay I generated a HeLa cell lines that expressed GFP-tagged fusion protein. The drug treatment was carried in 384 well plate for 16h and then analysed with a confocal microscopy meant with highthroughput-fluoroscence capability. for analysis I calculated the nuclear:Cytoplasm ratio of the GFP signal for all the drugs and the chose the top 8 drugs with the median N:C ratios.
For validating my screen I treated the same cells with the top selected drugs in 10cm plate, used the regular cellular fractionation techniques to separate nuclear and cytoplasm fractions and then checked my protein levels through western blotting. I see very less nuclear exprssion of my protein of interest while its largely present in the cytoplasm. This does not contrasts with my HTS results.
I hope you can now understand my strategy and the issues in it.
right, sorry - misunderstood. Nuclear/Cytoplasmic separation can be very problematic, as you have seen. Do you at least see separation of nuclear and cytoplasmic control proteins (e.g. Latin B vs Tubulin?), so you know that your fractionation is working? As possibly your nuclear membrane is getting damaged in the first step and you need to use less harsh detergents etc in the protocol...?
My fractionation is very clean. I have checked it with proper controls - HSP90 for cytoplasm and c-Jun for nuclear fraction. The protocol has been repeatedly used and works fine.
I am struggling to critically analyse it as both Western blots and HTS screen are right in their individual sense but to bring them together critically, I am finding it difficult.
Have you tried to repeat the original microscopic observation with the hits to make sure they were not mistaken? Is the drug material you are using for the fractionation experiment from the same supply as the original HTS hit?
Ya .. The only reason for producing the GFP tagged version of my protein of interest is that my protein is that we do not have a good antibody that can detect the endogenous protein. There is no change in the characteristics of the cells upon overexpression. My protein is expressed at very high levels in all cancer cells even at endogenous levels.
There are many levels of validation for HTS hits. Since HTS normally produces n=1 results for hits, the first phases of validation are replication, then dose response curves, using the original HTS assay conditions. Did your hits pass these tests? Have you proved that your secondary validation assay actually works reliably with positive controls? For any HTS project your downstream methodologies for validation need to be worked out flawlessly in advance, or you are wasting your time and money doing screening.
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