Questions tagged [assay-development]

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Why such strange enzyme kinetics?

I measured some enzyme kinetics in a practical course using a substrate-based FRET assay. Unfortunately some of my plots show weird effects. There was always a decrease in signal after 35 minutes. But ...
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What is the dynamic range under initial conditions?

could someone help me to understand the following sentence better? It's from the book "A Practical Guide to Assay Development and High-Throughput Screening in Drug Discovery". And it's about ...
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Antibody detection - infection-induced vs vaccine-induced - is it common (or not) for a test to be positive for both?

Given a pathogen and a corresponding vaccine, and given an immune response to either one that results in antibody creation, would it be expected that a clinical test or assay for said antibodies would ...
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Enzyme inhibitor leads to higher turnover rate?

I'm currently working on a project where I have to deal with enzyme inhibition. The purified enzyme shows a good substrate turnover. When I try to inhibit it with different inhibitors described in ...
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Why is thymidine incorporation (DPM) normalized to per mg protein content while testing for cell proliferation?

Sorry for this naive question. After performing the thymidine incorporation assay to test proliferation, many papers report normalization of disintegrations per minute (DPM) to per mg protein content? ...
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1 answer
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Decreasing signals in assay measurements

I'm working with a calcium assay to study the effects of different virulence factors. The assay works, but from day to day the signals of cell lysis go down. Unfortunately, I haven't found an ...
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Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
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3 votes
2 answers
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Finding total concentration of enzymes

Sorry if my question is very basic for biology majors because I am not. I am trying to build a mathematical model of a particular pathway using systems of differential equations and in order to reduce ...
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TMB vs ECL for ELISA: which detection method is more sensitive?

I failed to find a comparison, does anyone know which ELISA detection method is more sensitive of these two: ECL, TMB? Many thanks!
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Easy and cheap antigen/antibody couple

For an application I need to find a cheap antigen and cheap correspondent antibody. The antigen can be literally any molecule that is cheap and potentially easy to produce and with a correspondent ...
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How to choose an assay for detecting virulence factors?

When looking for a protein of interest, an ELISA is being administered. I read this article about the different types of ELISAs and the advantages/disadvantages. However, I am conflicted about ...
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2 answers
102 views

Using autoclaved store-bought distilled water for labwork?

I'm a high school student who's working on a molecular biology project in my school's lab. I need pure water for making culture media, buffers, running a BCA protein assay, running digests, cleaning ...
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Where can I find hemagglutination inhibition (HI) assay data?

I am looking for hemagglutination inhibition assay data for type A influenza virus. I've checked in databases such as fludb.com, however it seems to only have genetic data. A lot of the time, ...
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1 answer
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How can enzymes be immobilised on glass?

I’m studying a hypothetical model for urease activity, which involves fluorescence measurement, hence the need for an optical window to which the enzyme urease is immobilised. From my previous ...
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Can experiments in ELISA kits be monitored via both fluorometry and photometry?

In my bioanalytics course slides, the professor has written at one point that in a heterogenous immunoassay such as ELISA, we use fluorimetry to measure concentration of an analyte. In another slide ...
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Minimum number of cycles for effective qPCR quantification?

I have been having an apparent problem with gDNA contamination in my no-reverse transcriptase (NRT) controls by the appearance of fluorescence peaks in my qPCR data. I have/am trying multiple DNase ...
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Assays for protein or lipid content of insect pupae

Does anyone have any recommendations (based on personal experience) for rapid, cheap, accurate assays for estimating protein & lipid content of individual insect (mosquito) pupae? So far I've ...
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4 votes
2 answers
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Definition of a Katal (unit of enzyme activity)

I am very confused about what one 'Katal' actually is. From Wikipedia, "The katal is not used to express the rate of a reaction; that is expressed in units of concentration per second (or moles per ...
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Can you freeze an ATP assay sample for later use?

I am going to use adult Drosophila to do an ATP assay (ATP determination kit A22066). The most convenient for me is to freeze the samples and perform the experiment later. However, the protocol ...
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Which analytical techniques could you use to research relationships between 2 proteins? [closed]

Two of the proteins I am researching are known to interact. However, I would like to know if they interact with other proteins as well, and possibly form a pathway. Which analytical technique(s) I ...
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Why would you plate cells at different densities in a colormetric assay?

The aim of my experiment is to see if a kinase inhibitor reduces cancer cell viability. I am using 2 different cell densities 50,000 cells/well and 100,000 cells/well and different doses of the kinase ...
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2 votes
1 answer
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How do detergents interfere with protein assays?

This has been getting me stuck. I've tried to understand what a detergent would do in an assay, but I can't figure out whether it would affect the protein or the reagent (say, in a Bradford assay).
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Where am I going wrong in a Reactive Oxygen Species Assay done in N9 cells?

I have followed the modified protocol that was given to me by my senior (who was using for his RAW 264.7 cells - Reference article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308605/ ) for ...
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Measuring optical density with/without re-suspending cells

When measuring absorbance using a plate reader, is it necessary (or better) to resuspend the culture if the plate has been sitting still for 1-2 days? While this could be a good physics question, I ...
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Ways to monitor enzyme kinetics with very fast time resolution?

I'm interested in any way to do time-resolved study of enzyme kinetics. I am studying some physical variables that may affect kinetics, but I want to study how quickly they take effect, and how long ...
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T cell sensitivity and persistence to specific bacterial proteins

Currently, the standard tests for Lyme Disease measure antibody production after exposure to a bacteria, Borrelia burgdorferi. Often, the tests are performed too soon after infection, before ...
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3 votes
2 answers
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Can ELISA be used to detect a plant enzyme? Creating assay for a new enzyme

If the goal is to generate a rapid assay for an enzyme of plant source what are the typical options? i.e. Could one do something like: Generate an antibody to the enzyme and then use it to create an ...
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1 vote
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Assays to determine competitive binding versus non-competitive

I'm looking for both simple and complex assays or technologies than can be used to determine if two competing molecules are competitive or noncompetitive. I figure xray crystallography is a clear one,...
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How to calculate the LOB, LOD and LOQ of an enzyme assay

I understand how to calculate limit of blank (LOB), limit of detection (LOD), and limit of quantitation (LOQ) in the traditional way i.e., average and SD of raw analytical signal of blanks and low ...
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1 answer
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Is galvinoxyl antioxidant assay possible using NMR spectroscopy?

I would like to perform an antioxidant assay using the galvinoxyl protocol. The protocol states that we need EPR spectroscopy, but only NMR spectroscopy is available at my institution. Is there an ...
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1 vote
1 answer
37 views

Colormetric assay for a phenol product?

I am working on an assay for phenol products created by a colony of bacteria. I have searched up a paper that involves gibbs reageant but the protocol is kind of unclear. Does any of you know where I ...
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2 votes
1 answer
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Is NADPH unstable in UV light?

I am working on an enzyme activity assay using NADPH as a cofactor. The MSDS for NADPH Tetrasodium Salt, tells to store it in a place away from heat and light. Does this include UV light or just ...
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1 vote
1 answer
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Choosing the best assay for my experiment? [closed]

Which of the following assays can be used to determine whether cell death specifically due to apoptosis has occurred in a given cell sample? Choose all that apply. WST-1 Assay Caspase Colorimetric ...
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Background correction when reading ELISA with TMB substrate

I'm doing some ELISA development, and I'd like some justification/best practices for background correction. I'm using a horseradish-peroxidase (HRP) detection system along with a TMB substrate and an ...
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What is the probe that absorbs at 450nm in the presence of NADH in this assay?

The colorimetric assays by Biovision and Sigma Aldrich seem to utilise a probe that binds to or reacts with NADH in order to cause absorbance at 450nm which can then be quantified by a ...
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2 votes
1 answer
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Pectinase Enzyme Assay

I am working on pectinase enzyme assay. I incubated 900 ul of substrate for 10 minutes in the water bath, followed by adding 2ml of DNSA reagent, then 100ul of enzyme extract added finally i read the ...
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2 votes
2 answers
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Assays during drug discovery

After researching the definition of Assay, I am left with the idea that an assay refers to scientific screening. It could be of chemicals, microbes, etc. I understand that during drug-discovery ...
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2 votes
1 answer
4k views

What would be the best design for spike-and-recovery and linearity-of-dilution validation experiments in one 96-well ELISA plate?

I have searched in the web for a detailed explanation of doing such validation experiment, but unfortunately couldn't find a satisfactory one. I came across the following sources: Thermo Scientific ...
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6 votes
1 answer
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Primary cilia: what cell types have non-motile cilia that migrate?

My understanding is that there are two broad categories of cilia: motile and non-motile (also called primary. Examples of the former include sperm flagella and the cilia of epithelial cells that ...
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7 votes
1 answer
109 views

Providing small molecules to cells on a filter plate

Lets imagine that I have mammalian cells that I've immobilized on a filter. Now I want to keep providing small molecules to these immobilized cells without resolubilizing the cells. The caveat is ...
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5 votes
0 answers
59 views

Serological assays not detecting native proteins [closed]

Is there anyone out there who has done much work with serological assays? We have antiserum for a manufactured viral protein but no luck so far getting it to detect native protein (unless today's ...
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6 votes
1 answer
148 views

Ideal low-protein binding membranes

I'm looking through the literature on the topic. It seems like hydrophilic PVDF membranes are ideal for low-protein binding but it also sounds like Regenerated Cellulose is an appropriate substitution....
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