High-Throughput Screening Assays - Science method
Rapid methods of measuring the effects of an agent in a biological or chemical assay. The assay usually involves some form of automation or a way to conduct multiple assays at the same time using sample arrays.
Questions related to High-Throughput Screening Assays
Hello, I am intending to screen a library of compounds for an in vitro enzymatic assay. The target enzyme follows a Bi-Bi mechanism. The assay development phase is completed with kinetics parameters for both substrates are determined. I am looking into a rational approach to choose the concentration of both substrates and compounds to satisfy the following criteria:
-Inhibition Mechanism-blind design: the mode of inhibition and the substrate of which the inhibitor compete with is unknown.
-Minimize the concentration of the compounds to a reasonable concentration that still able to pick up any possible inhibitory mechanism.
Successful compounds will be used to identify Hits with dose response and IC50 later on.
Is there any recommendations/guideline followed in pharma to deal with this? is there any upper limit for a Hit to be accepted?
We want to develop a screening test based on the measurement of GALC enzyme activity in patient fibroblasts (Krabbe disease) like the one described in this paper ( Ribbens, J. et al. A high-throughput screening assay using Krabbe disease patient cells. Anal. Biochem. 434, 15–25 (2013)) with the use of the substrate : 6-Hexadecanoylamino-4-methylumbelliferyl-b-D-galactopyranoside.
Unless I am mistaken, there is no mention of the standardization technique in this paper.
We know that the molecules we test can have an effect on cell growth and therefore on the final amount of enzyme during the enzymatic reaction.
What is the best way to standardize this type of cellular enzymatic test which is HTS compatible..?
We hope to produce a recombinant mycobacterial RNA polymerase through heterologous expression in E. coli to evaluate the activity of some drug candidates as RNAP inhibitors.
Unfortunately, up until now, we have only found references that propose methods for High Throughput Screening (HTS) and that is not our goal.
Does anyone know a method that could be applied in a smaller scale, preferably that does not include using radioactive reagents?
As a part of my MSc by Research we will be utilising high throughput microplate assays following Jackson et al. (2013), a technique that utilises 4-methylumbelliferone (MUF) labled substrates.
In our lab we have some of the necessary model substrates ( 4-MUF-β-D-glucopyranoside and 4-MUF-N-acetyl-β-D-glucosaminide) but their condition is unknown.
Does anybody know if there is a way to determine if they are still fit for purpose as they are expensive to purchase and it seems like a waste of money to purchase more if they are still good to use.
Thanks in advance for the help.
I'm planning to start designing an in vivo assay to be used for a HTS. Does anyone have any advice/know of literature on the subject on how to choose an appropriate cells line? I will have to establish a stably transfected cells line with at least two constructs. The readout will be measuring changes in GFP expression.
Potassium acetate is a good buffer used in high throughput screening for cellulose-degrading enzymes, can we replace it with sodium acetate or other buffer?
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?
Need a contract lab for high through output assays.
Please share leads and intros [email removed by admin]
US based preferred.
I am trying to choose a good human cell line for my screening assay.The cells will undergo virus transfection and sort the positive cells and then transfection with plasmids after that sort again.So I need to know which cell line has high efficiency and easy to culture.
We are using Nunc™ 96-Well UV Microplates for measuring BSA protein concentration. It is a little surprise that the standard curve is quite different comparing to the one measured by the spectrometer at 280nm. The slope of the standard curve obtained by the plate is way off the value of extinction coefficient for BSA .
I have developed a cell-based assay in 96-well plates. After stimulation of mouse neuroblastoma N2a cells with a growth factor I fix them with methanol and measure phosphorylation of Erk1/2 (staining with phospho-antibody against Erk1/2 + fluorescent secondary Ab). The intensity of fluorescent signal is measured in a plate reader. Because I need to use these cells for an automated high throughput screening, I had to switch to the 384-well plate format. Unfortunately, cells detach from the bottom of the plates during washing steps (automatic washer is already set for low speed dispense and aspiration). It has never happened in 96-well plates - the cells stayed attached during all the procedures. My guess is that in smaller wells (384-well plates), the force of meniscus works against cell attachment so they are not as firmly attached as in bigger wells (96-well plates). I only tried Greiner's tissue culture (TC)- and Advanced TC-treated plates. Perhaps I should try other vendors/plastic surface treatments that would warrant the firmest attachment of my cells. Anyone else has the same problem? I would appreciate your suggestions. Thanks!
My purpose to screen some compounds interacting with membrane receptors.
I want to check the proliferative effects of some anticancer drugs (more than 50). I observe through CCK-8 or MTT assay. What is the best assay to check their proliferative properties other than MTT.
Does anyone have any experience with Luminex screening assays? How reliable are the assays (precision and selectivity) when compared to running individual ELISA assays?
I am having trouble distinguishing an unhatched/dead C. elegans embryo from a remaining egg-shell of a hatched embryo in a bright-field image in a high-throughput fitness assay.
I was wondering if one could discriminate between an unhatched/dead embryo and the remaining chitin shell by adding some dye to it. DNA dyes (Hoechst 33342, 33258, Sytox green) are not penetrating the embryo. Chitin dyes (Calcofluor white, congo red) are just staining the chitin shell of a hatched and an unhatched embryo in a similar fashion.
I would be happy if someone would provide an idea.
I have a library of tagged proteins overexpressing cells and I have total cellular extracts for all of them but in small amount (around 500 ng of total proteins for each) and I would like to avoid defreezing the cells library to regrow them and reharvest largest protein samples. I would like to set up high throughput interaction assay of all these guys with a single protein. I thought about Gst-pulldowns, ELISA or Alpha screen technology. Also the protein samples are conserved in -20C, can the freezing alter their capacity to interact? I am looking for the fastest reliable technique.
I need a screening method to screen apoptosis in cell cultures. I have an idea that could be interesting but I need some preliminary data to decide whether it make sense to ask my PI to invest on my idea or not. My lab is specialized in biochemistry, molecular biology and proteomics so I have all I need to perform Western Blot, ELISA or Real Time PCR. I also do have a spectrofluorimeter but it's not a 96 well plate reader. For the microscope... well, let's assume I don't have one, because the one I have it is barely enough for cell counts :D
If my idea proves right I can ask to use other's laboratory equipments but I first need some preliminary data!
I tried an ssDNA ELISA kit but it was really terrible (the kit itself, not the data).
Any other suggestions?
Hi, I usually work with 384 well microplate (square well with flat bottom) for absorbance measurements. In some cases, particularly with bradford reagent for protein determination, the O.D replicates measurements are sometimes bad. I've realized that the shape of the meniscus is not formed correctly, probably due to the composition of reagent and its interaction forces with the square plastic well. Agitation of the plate can decrease the replicate variability but it doesn't give entire satisfaction.
I think that a good way to solve this problem should be the use of 384 well microplate with round wells (with flat bottom). If anyone knows a reference supplier it would be great.
Why would someone choose to perform siRNA HTP over small molecule inhibitors HTP or vice versa? If you perform an siRNA screen and end up finding a target and then develop a small molecule inhibitor for that target, then why not from the beginning screens for a small molecule inhibitors? Are there any major advantages or disadvantages of performing one over another?
I would like to screen through hundreds of colonies for cell growth. Is there an easy way to grow hundreds of different cultures of e.coli in a shaker incubator with limited space?
Ideally there will be at least a half a ml per sample, and I will be able to measure OD simply and quickly.
Price isn't much of an issue.
We recently published an article, “Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses,” Ekins, Olechno, Williams, PLoS ONE 05/2013; 8(5):62325. In it we show strong evidence that the manner of transferring fluids and the methods used in managing dilutions can dramatically affect the results of your assays. The article was published in PLOS ONE, an open access journal. We want to know whether other researchers have experienced similar results. We would like to hear questions and comments about the paper.
I would like help with reproducibility in biofilm quantity and less time consuming (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species).