High-Throughput Screening Assays - Science method

In my experience, the usual concentration of test compounds from a generic compound library used in high-throughput screening to search for enzyme inhibitors in an in vitro assay is 10 µM. For fragment libraries, a higher concentration, such as 100 µM may be used because the molecules are smaller and simpler, overall, than the molecules in generic screening libraries.

Going to higher compound concentrations may seem appealing, but the result is not necessarily better because you run into increasing problems with compound insolubility and interference with the assay readout as you increase the compound concentration.

As for the substrate concentrations, there is no "right" answer. There are trade-offs. Since you know the kinetic mechanism and the substrate kinetic parameters, you can calculate the ratio IC50/Ki using the appropriate Cheng-Prusoff relationship for inhibitors competitive with one substrate or the other as you vary the substrate concentrations. This ratio can be used as a measure of the sensitivity of the assay to find inhibitors. (For pure non-competitive inhibitors, the substrate concentrations don't affect the sensitivity.) I suggest you prepare a spreadsheet with columns for [A], [B] and IC50/Ki. Vary A and B and calculate IC50/Ki.

Typically, the lower you make one substrate concentration, the more sensitive the assay is for being inhibited by compounds competitive with that substrate (lower IC50/Ki), but the less sensitive it is for finding inhibitors competitive with the other. You can try to find a pair of substrate concentrations that achieve a balance.

To get around this issue, you could run two separate screens, if you can afford to, with one optimized for finding inhibitors competitive with A and the other optimized for finding inhibitors competitive with B.

You also have to consider the substrate concentrations needed to get a sufficient signal to run the screen successfully. Higher substrate concentrations allow a larger signal to be obtained while still under initial rate conditions, but higher substrate concentrations increase IC50/Ki. Cost and/or substrate availability may also have to be considered.

Kindly check https://www.sciencedirect.com/science/article/pii/S2213020914000032

If you are going to use purified RNA polymerase, then you can measure the production of pyrophosphate that results from RNA polymerization using a plasmid and 4 ribonucleotides as the substrate. A colorimetric method for doing this assay with E. coli RNA polymerase is given in this paper:

You should include a plasmid-free control because there could be some background nucleotide hydrolysis in the absence of the plasmid.

The answers above are great, and I agree that your compounds are likely still good for use.

Still: Buy new ones.

The time you/your student work on establishing that they're still good will cost more that re-ordering the labelled substrates, and it's one less issue that could come up during review when you get your results published.

The attached article could be of help ... but maybe too late ...

Best regards

Robert

Dear Farah,

Yes, you can. For more details, please see attached publication (Figure 2):

Kračun et al. Biotechnology for Biofuels (2015) 8:70

DOI 10.1186/s13068-015-0250-y

A new generation of versatile chromogenic

substrates for high-throughput analysis of

biomass-degrading enzymes

Stjepan Krešimir Kračun1†

, Julia Schückel1†

, Bjørge Westereng1,2,3, Lisbeth Garbrecht Thygesen3

,

Rune Nygaard Monrad4

, Vincent G H Eijsink2 and William George Tycho Willats1*

Abstract

Background: Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.

Results: We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates

of biomass materials in which the complexity of plant cell walls is partially maintained.

Conclusions: We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic

enzyme screening systems.

Keywords: Chromogenic substrates, Carbohydrate-active enzymes, High-throughput screening, Biomass degradation, Plant cell walls, Lytic polysaccharide monooxygenases

Hoping this will be helpful,

Rafik

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. 

Hello Douglas,

Our lab, the Integrated Microscopy Core at Baylor Houston, does high throughput screening and assay development. We have available a BioMek Span8 robot for plate processing and Vala IC-200 high throughput microscope. We are a Core facility and can accept samples from anyone, anywhere. Can you tell me more about your research? Please visit our web site for more information.                  

PA319 or NIH3T3 would be good.  Check your cell culture protocol.  293 works well.  If you have problem with it, then obtain 293 from company instead of from other lab.

You have to consider the path length through the solution, since the absorbance is directly proportional to the path length (see Beer's Law). For a spectrophotometer cuvette, the path length is usually 1 cm. In a microplate, the path length is proportional to the sample volume.

I was quite unhappy with Greiner plates. You could try BD or sarsted

It all depends on your screen. As mentioned above, luciferase tends to be more sensitive and have lower background and the time needed to scan each plate on a plate reader is very short. However, it is time sensitive and you need to add the substrate to each assay plate. If you have access to a plate reader that will automatically add the substrate for you this is not a problem. If you have to add substrate by hand, it takes time and will limit the number of plates you can assay at a time. It also can be expensive if you are doing a large screen. The costs and time of fluorescence depend on your setup. If you have a fluorescent reporter protein that can be read out on a plate reader (all you need to read is intensity) it can be quick and cost effective. Sensitivity tends to be a bit lower than for luciferase but if you have a good reporter and a sensitive fluorescent plate reader it may be a good choice for a primary screen. Than you can follow up with a more quantitative secondary screen using luciferase. If you need to do immunostaining and than image your cells it will be more expensive and slower. You will also factor in time and know-how needed for image analysis. But you will get way more information.

The best way is to assay both cytotoxicity and viability. For cytotoxicity, measure of LDH release is a straightforward fluorescent assay, amenable to medium throughput: it measures the amount of dead cells after a chronic or acute insult. There several kits available in the market that make your life easier, but it is also simple to make the assay reagents in house. For viability, I would choose ATP production. There are also several kits in the market easy to optimize, such as ATPlite.

Hi all, I plan to use this luminex screening assay as well however I was wondering if the kit can be use to detect cytokine in body fluid? My sample is bronchoalveolar lavage fluid. Anyone can help?

I think vital stains (so called because it does not harm the embryonic cells) like Nile blue and neutral red  can do the job effectively. They can be absorbed in bits of agar and applied on various positions of surface of the embryo. The dye gets inside the cells and marks them throughout development. The German embryologist Vogt was the first to use this method (1925) to show that each stained patch gave rise a certain part of the embryo.

You could give it a try with MST (MircoScale Thermophoresis). To do so, you would need to label your interacting protein. Then you fill each of your cell extracts into a reaction tube (or in a plate) and add the labeled protein. The reaction setups are filled into glass capillaries, and the measurement can be started.

During the measurement, a temperature gradient is induced which makes the molecules move (Thermophoresis). Their movement depends on different parameters that typically change when your molecule of interest (in this case your labeled protein) binds to an interaction partner (in your case this would be the overexpressed protein in the cell extract). You also need to perform negative controls with control cell extract (no protein overexpression) and in the best case scenario you also have a positive control.

In case you are not only interested if the two proteins are binding to each other, but you would also like to determine the affinity, you need to quantify the overexpressed protein. Then you need to prepare dilution series of the cell extracts. The change in thermophoresis is plotted against the ligand concentration; fitting the data points you will obtain the binding affinity.

Sure, freezing can change protein properties, depending on the freezing conditions (volume, speed of freezing, storage temperature, addition of glycerol etc.). We have best experience flash-freezing small aliquots (10µl) of pure protein in liquid nitrogen. For cell extracts, I guess this could be a different story.

If your cells follow the canonical pathway and display membrane Annexin, there are colorimetric based ELISAs that you can purchase or make.  

Thank's Graeme !

I'm looking for clear microplates and the Corning are white or black only. I've searched on their website but still nothing...

First to be sure to get the 100% inhibition both siRNA and HTA is used. Secondly si RNA inhibit by binding directly to gene thus stoping transcription of gene of choice subsequently translation, while most the HTA or other chemicals bind mostly to protein that subsequently inhibit either transcription or translation. I think this could be possible answer of your query.

Thanks.

For that volume of cells, this can be done in deep-well 96-well plates with shaking. It would require transferring cultures via multichannel pipette into 96-well microplate for OD readings on a plate reader. Alternatively, if you only need OD values and have access to plate readers with incubator/heating and shaking functions (fairly standard on many readers), you can take realtime growth measurements. This allows you to automate the process and take as many OD readings as you would like. Hope that helps.

Liquid handling settings most definitely affect assay quality as measured by z’ factor or %CV. This includes accuracy and preciseness of liquid transfer, which affects the biological response to given controls and test compounds. Furthermore, if the assay utilizes attached cell lines, then the dispense height, dispense speed, and mixing speed and depth affect attachment of the cell monolayer. If the dispense height is too close to the monolayer or the dispense speed is too fast, the cells can become detached which will negatively affect assay results. All of these parameters are part of assay development, and have to be fully optimized. Additionally, different cell lines have different attachment strengths so these parameters have to be optimized to each cell line that is utilized.

Dear Karthikeyan the most reproducible antibiofilm assay is the MBEC Assay System produced by innovatech. With best regards