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Freeze-thaw cycles are often suspected of causing degradation in organic molecules 1,2. My first guess would be that your fluorophore is breaking down due to those repeated cycles. Alternatively, you might be getting precipitation of a calcium compound from the buffer you are freezing that your colleague is not. Standard laboratory practice is to make ...


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Glass can be functionalized by organosilanization so that biomolecules can be covalently attached via some cross-linker. As an example, one might aminosilanize the glass and then cross-link their enzyme of interest with glutaraldehyde. For what it’s worth, urease activity can be measured in solution.


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I'm a pharmacy student and I can try to explain how they do to find new drugs. In fact, there are many diferent ways to do so, it depends of what kind of new drug you are searching for. Sometimes you're working on a disease which the pathological molecular mechanism of action are well-known and where many drugs are already effective. In this case, the '...


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An assay may be thought of as a trial or test that is designed to quantitatively determine the amount of a substance in a sample. Thus a biochemist might use a protein assay to quantitatively determine the amount of protein in a sample, or a pharmacologist might assay a sample to quantitatively determine the amount of drug present, or an enzymologist ...


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Yes, distilled water would be absolutely fine for your needs. You really don't need ultrapure water except for the most sensitive of applications. You also don't need to autoclave the distilled water before making things like media/buffer/etc; instead you would autoclave/filter them after you make them. You can take a look at this document for an overview ...


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I want to add something on top of Chris answer. The production of an antibody it is usually a quite slow (and expensive) process, an alternative that worth to consider is phage display (https://en.wikipedia.org/wiki/Phage_display). Once you find the phage that effectively bind your protein of interest, you can use it instead of an antibody in what is called ...


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I think the most promising routes use antibodies. You could either develop an ELISA or do western blot analysis of plant material - both need a good and specific antibody. To generate these, the protein of interest (or at least parts of it) are injected into animals (typically mice or rabbit) and then antibodies are pourified from the blood of these animals. ...


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Galvinoxyl is a free radical, which means it has an unpaired electron that can be detected with EPR. Antioxidants reduce the radical, which is then not detectable in the EPR anymore as it doesn't have an unpaired electron. What you need the EPR for in the assay is to determine how much of the galvinoxyl is still a radical. Paramagnetic molecules like ...


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The comments you have given are some reasons, although they also depend on the confluency of the cells. Your teacher/professor/advisor is also making sure that you can see an effect at low levels of drug, as well as making sure that changes in the signal are visible - for example, does a 50% reduction in viability due to drug activity give the same drop in ...


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I don't think there's any universal database containing ongoing, widely representative HI assay output. The Antigenic Cartography group has made available some historical datasets that were used in published articles, such as Influenza A/H3N2 data published in Smith et al. 2004. Science 305:371-376 Data from H1N1pdm09 assays in 2009/2010 (click on the ...


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In general, ECL is more sensitive than TMB for ELISA detection. The minimum detection limit of TMB is 60pg/mL and 20pg/mL for ultra TMB [1], whereas the detection limit of ECL can be as low as about 1.7pg/mL [2]. Resources: https://info.gbiosciences.com/blog/elisa-substrates-a-selection-guide https://www.thermofisher.cn/cn/zh/home/life-science/antibodies/...


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This is done because at some point you start amplifying nonsense and not get a meaningful signal. If (and this is most often not the case) you have the ideal doubling of you DNA template in each cycle, you can calculate how little DNA you start with to detect it only around cycle 40 - or the other way round: What you will amplify and see as a "signal&...


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Short answer: no. Because all vaccines (that I know of) rely on protein or polysaccharide production in some form, whether "natural" (e.g. inactivated virion, subunit vaccines) or "artificial" (purified protein from expressed genes) forms, and the aim is to elicit an immune response that targets the natural pathogen form, so you need to ...


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While it's difficult to know just what is going wrong in somebody else's laboratory, apparently paradoxical effects like these are unfortunately common in laboratory work. Assuming that these are indeed well-established inhibitors that really should be working, let's consider why they might not be in your hands. The mechanisms of molecular interaction are ...


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I'm not sure that there is a strongly disciplined reason for the mg protein content as the normalization, but there is obviously a need for some normalization for different amounts of tissue- if you use 10X as much tissue, you should expect DPM to be 10X as high! For example, in this somewhat old paper, They normalize not to protein content but to wet tissue ...


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Hi again TheLast Cipher, Enzymes in a cell are usually semi-quantified by the: Western Blot technique. But there are lesser known techniques. E.g. For a Western Blot the unit of measurement is the color intensity of the enzyme band on the blot membrane. If I were you I would look for journal articles covering your pathway of interest. E.g. https://iubmb....


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A good choice is the IgG/anti-IgG pair for cheap, easy to demonstrate/work with system. E.g. Goat-IgG coupled with donkey anti-goat IgG. Just about any antibody supplier will have these. These type of systems are the basic primary/secondary antibody bindings routinely used in immuno-assays, blotting etc. These may be mouse IgG coupled with a labeled anti-...


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can we use both methods depending on whether the marker enzyme on the antibody is given a fluorescent or chromogenic substrate? This is essentially correct. You can have the same ELISA work on different methods of detection. The most common ELISA is colorimetric, and works by binding a secondary antibody conjugated to some enzyme to your primary antibody. ...


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Although this is old news, as @R.M. has been good enough to provide an answer, I feel I should add my own. The reason is not that I think his answer is incorrect in any way — it is technically quite correct and I am not going to repeat or rephrase his technical arguments — but I do not think it addresses what I regard as the misunderstanding in the poster’s ...


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A katal is indeed a specific amount of enzyme (rather than a reaction rate), but one which is measured with respect to the catalytic activity of the enzyme, rather than with respect to the number of molecules, their weight, or their size. Roughly speaking, one katal is the amount of of enzyme which can convert 1 mole in 1 second. (Just like one pascal is ...


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Pure ATP can be stable when stored frozen but I would be worried, perhaps unjustifiably, about storing a homogenized sample. You should try dividing a homogenized sample in half and assaying one half right away and the other half after freezing to see how the signals compare.


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There are plenty of methods to use. Here is a overview and background of some common methods as written by ThermoFisher: https://www.thermofisher.com/se/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-protein-interaction-analysis.html There is also phage display ...


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Depending on the detergent, its concentration, and the exact assay being performed, it can affect both the protein and the assay reagent(s). Some detergents will bind the (usually colorimetric) reagent, or otherwise chemically react with it, giving high background to your assay and sometimes completely masking the specific signal of the assay itself. Many ...


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Definitely (2). Clumps of cells will cause a lot of noise in your absorbance measurements (depending on whether the light beam hits a clump or some empty space). I don't know what your microbe is or what growth medium you're assaying on, but typically it's very important to maintain the culture well-mixed at all times, not just during measurement. If your ...


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Some ATPases can work with MANT-ATP or similar fluorescent ATP analogs that change their fluorescence properties upon protein binding as well as hydrolysis of their phosphate group. This has been used frequently (see here or here) to study enzyme kinetics on fast timescales.


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I have not used the assay myself, but Bhuiya and Liu "A cost-effective colorimetric assay for phenolic O-methyltransferases and characterization of caffeate 3-O-methyltransferases from Populus trichocarpa." Anal Biochem. 2009 384(1):151-8 [link] seems to have a relatively straightforward protocol. Roughly, you make a 0.4% (w/v) solution of Gibbs reagent in ...


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In my experience, reuse of NADPH stock solutions maintained at pH 7.0 or above is perfectly acceptable. Taking an absorbance spectrum every half-hour or so will probably show that definitively. However, NADPH is VERY unstable in acid, leading to a 'bleaching' of the absorbance at 340nm. Try measuring a rate at pH 6 or below, 340nm, with 100 micromolar ...


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Using different wavelength for reference give some advantages. You can measure a baseline of each well. All well might not have the same baseline because each well might not be the exactly same as others. In addition, you could make scratches on it, you might touch the bottom, or you get some dirt from your bench. And this can be corrected by A540 in your ...


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So, your blank is everything but the enzyme? That is, substrate and DNSA with 100 uL water? If yes, measure, as negative controls, substrate alone and DNSA alone. If one of them is also highly absorbing light, it may be contaminated, and a new vial should be ordered. If both are highly absorbing, it may be the water you are using, or the spectrophotometer. ...


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It's hypothesized that planar cell polarity is many cases functionally associated with asymmetry of (primary) cilia (Axelrod (2008)). So, the phenomenon of primary cilium migration could be not that infrequent. One example is the development of ependymal cells in brain ventricules: They are multiciliated and the cilia are motile, but ciliogenesis starts from ...


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