I want to detect kinetic of beta amyloid aggregation in present of lysed cell of caco2 cell line and in present of beta amyloid? please help me
Hi,
since you posted a question about advise on how to design an experiment it would be helpful to understand a bit better the broader questions you are trying to answer, but I`ll do my best with what I have. My guess is that you suspect something in this lysate will alter the aggregation process and/or kinetics. I have done similar experiments and they were straightforward. You mix Abeta with ThT and the substance which is expected to modify the aggregation and compare the time dependent ThT signal to a control with only Abeta and ThT. You can record the fluorescence in real time in a cuvette (also you might have some sedimentation effects of larger aggregates that will make your solution inhomogeneous, which can be prevented by stirring the solution, but this leads to other problems like sheer forces on the aggregates, creating air bubbles that scatter light and similar things that aren´t necessarily reproducable) or you can take samples in regular time intervals of your aggregating sample (after mixing it well) to reduce the sedimentation effects.
Where it gets messy is if you add the whole lysate. So if you have any idea what it is about the lysate that possibly modifies the aggregation kinetics, I would recommend to isolate it so that you will have a better defined system. If you add the whole lysate there are so many players that can interfere with the aggregation and the ThT-fluorescence and of course also the physical properties of the solution would change (the pH you can easily adjust in your control sample, but also the thermodynamic activity of water would change and you would have a vast number of interaction surfaces your key players are exposed to). If you can´t isolate your key component in the lysate or at least fractionate it, my two major concerns you should at least check for would be:
1. ThT can in my experience interact with many things. Everything that restricts the rotation of the two ringsystems increases its quantum yield. I guess if your starting fluorescence (of course with the same dye concentration) is the same you can assume that ThT isn´t bound to anything randomly present in your lysate (also you can´t be 100% certain.
2. The Abeta aggregation might already be altered just by the presence of other substances in solution. Higher crowding and availability of hydrophobic surfaces might push the protein to the aggregated state and therefore enhance the kinetic. This is difficult to correct for, so if you want to check for a specific effect of one interaction it is hard to separate these effects.
2.
Dear Christoph Röthlein
greeting
Thanks for your good answer. There are IDE (Insulin Degrading Enzyme) in caco2 lysis and I am using from caco2 as a source of IDE enzyme. I designed some peptide and want to assay the effects of them in IDE (IDE degrade monomer of beta amyloid so that aggregated beta amyloid will reduce after using IDE activator). But unfortunately I can not do it.
Is it possible that you exactly explain your experiment for me? or guild me?
I am looking forward you as soon as possible.
sincerely
my experimental setup was a bit simpler and as I explained, if you had a protocol to isolate IDE in its active form I would do that before I add Abeta and ThT, but from the way I understood it so far you have already tried your experiment and I guess with all the lysate, so here are some questions I would need to better understand, where your approach went wrong. Here are the three potential problems I would differentiate:
1. beta-amyloid does not aggregate in the presence of the lysate!
2. you see no change of fluorescence during the aggregation process!
3. The fluorescence changes equally with passing time in the presence and absence of lysate
Since I don´t know where it exactly went wrong, I 'll give you my quick analysis of all three cases and if you need more help please tell me a bit more about the situation:
1. in this case the concentration might be too low, if it aggregates in the same concentration without the lysate, probably the lysate stops the aggregation completely. In that case you are either lucky and the degradation via IDE works, or Abeta gets degraded by another protein in the lysate or it is just kept from aggregating by chaperones or other interactions to something in the lysate. In that case you will have to specify that better, which again would be a lot easier if you can isolate IDE from the lysate(in the functional form of course).
2. In this case it is likely that the lysate is too crowded to have a lot of ThT in solution free of any deactivating interactions in the first place. A dilution of the sample would help, but of course you would also dilute the concentration and effectiveness of IDE and the concentration of Abeta, which would slow down the aggregation process (which you could correct for by diluting Abeta equally in your control, but at low concentration the aggregation might really slow down or not happen at all.
3. In this case one possibility is that the IDE just can´t stop the aggregation process, maybe Abeta aggregates too fast and IDE can´t do anything to Abeta in the aggregated state (I assume you have the knowledge, that it can degrade Abeta under normal conditions, right?). Another possiblity is that IDE needs certain conditions like pH and ion concentrations that are not provided in this experiment) or maybe if you create the lysate yourself you set free some proteolytic enzyme that destroys IDE? Do you have a standard substrat with which you can make measure the enzyme activity? If it is inactive or slow compared to the aggregation process it will not have a significant effect on the results. You can slow down the aggregation by reducing the concentration of Abeta, so IDE has more time to interfere.
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