Science topic

Amyloid - Science topic

A fibrous protein complex that consists of proteins folded into a specific cross beta-pleated sheet structure. This fibrillar structure has been found as an alternative folding pattern for a variety of functional proteins. Deposits of amyloid in the form of AMYLOID PLAQUES are associated with a variety of degenerative diseases. The amyloid structure has also been found in a number of functional proteins that are unrelated to disease.
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I have been trying to purify Amyloid Beta 42 in my lab but this peptide is forming aggregates and I need this peptide in monomer form for fibrillation assay. I have gone through many papers where they have dissolved peptide in HFIP buffer but no-one has mentioned the concentration of HFIP buffer in which peptide was dissolved. It would be of great help if someone let me know.
Thanking you in advance.
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Thank you Nancy Pamela Aboytes Flores for your response.
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I am working on primary hippocampus neurons from mice. I am trying to look at the effect of amyloid beta on the neurons. I am looking at parameters like cell death, number of neurons in a field of view, neuronal length. I also want to try to analyze the neuronal coverage or area occupied by neurites to understand how amyloid beta affects the neurons.
Does anyone know how to measure the neuronal coverage or area occupied by neurites?
Thank You.
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The neuronal culture can be either live or fixed but would prefer to determine the coverage of neurites in live conditions.
Thanks in advance.
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I need to know which type of amyloid beta protein (human or rat/mouse) can be injected in mouse through ICV surgery for induction of Alzheimer's like phenotype.
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We are currently using congo red for staining amyloid in FFPE human tissue - the protocol is fairly simple, it involved deparaffinisation, antigen retrieval at pH6, incubation with 75% formic acid and then following the abcam protocol (this just involved hematoxylin staining, bluing, congo red for 20 minutes followed by a dip in ethanol and then histoclear).
What could possibly be going wrong? The samples are thinner than usual with congo red staining of only ~4um, however there appears to be absolutely no staining at all.
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The Abcam Protocol doesn't contain an antigen-retrieval. So I would stay to the original protocol. In my lab we do the counterstain with hemalaun after the congored. You can try this sequence and compare the results.
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I would like to ask one crucial question.
When I monitor Amyloid Beta (AB-42) concentration in cerebrospinal fluid (CSF), I get some values in lower and higher concentraitons. AB-42 concentration range in CSF varies between 200 - 1000 pg/mL according to the literature.
My question is following.
Do higher concentrations or lower concentrations of AB-42 in CSF indicate dementia ?
Thank you all for your answers or comments.
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Lower concentrations of amyloid beta 42 are associated with dementia
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I am currently doing aggregation Thioflavin T assays of my amyloid beta 1-42 and I am running into the following problem. 1. The fluorescence does not start at 0. 2. The fluorescence even decreases within the first hours. I used water and an aggregation Buffer (Tris pH 7.5, NaCl, EDTA, DTT, Heparin sodium salt) for the dilution. The amyloid-beta is dissolved in PBS and NH4OH, which should prevent aggregation. After adding amyloid-beta and ThT to the buffer/water, I immediately start the measurement. And the control (Buffer/Water +ThT) has already been subtracted from the values. Can someone help me ?
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Hi @Christina Sauerland,
have you pre-heated the plate reader that you start at 37°C right away? Also, have you tried using a different aggregation buffer e.g. PBS? This is usually sufficient to trigger the aggregation very efficiently from the start onwards, no Heparin etc. needed.
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I am trying to Generate polyclonal antibodies towards the amyloid-like species of glucosylcerebrocide (GlcCer). Rabbit antisera were collected and used against this GlcCer. I have prepared GlcCer amyloid sample in PBS. Now I am trying to find out the binding affinity of this rabbit antibody with GlcCer sample through DOT Blot assay. My experimental design is as follows:
Control lane: PBS+ rabbit antisera
Sample: PBS+GlcCer+rabbit Antisera
I found the same signal from both lanes. How do I optimize this error?
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Thank You Ellen S Vitetta.
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I am trying to Generate polyclonal antibodies towards the amyloid-like species of glucosylcerebrocide (GlcCer). Rabbit antisera were collected and used against this GlcCer. I have prepared GlcCer amyloid sample in PBS. Now I am trying to find out the binding affinity of this rabbit antibody with GlcCer sample through DOT Blot assay. My experimental design is as follows:
Control lane: PBS+ rabbit antisera
Sample: PBS+GlcCer+rabbit Antisera
I found the same signal from both lanes. How do I optimize this error?
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Try adding 0.1 or 0.2% m/v Tween 20 to all solutions but what you are using for coating. Also do checkerboard dilutions of antigen and antibody and determine the optimal dilution of the anti rabbit HRP first (how much may you dilute it before you see background?).
If you have access to ELISA-plates and a microplate reader, this approach might give you semi-quantitative data.
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We add amyloid β 1-42 to cells in order to create a model of Alzheimer's in cell culture. However, how can we determine exactly when Alzheimer's occurs in cells? Is there a method for this?
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Dear Beyza,
First of all, there will be not Alzheimer's disease (AD) occuring in your cells. You generate a cellular model for particular aspects of a complex disease, but your cells cannot get the disease in its full form...
Mario mentioned some aspects, such as cell death, mitochondtial integrity and gene expression.
One other typical aspect of AD is hyperphosphorylation of the microtube-associated protein Tau. You could check this using immuno-staining.
Another aspect is, that amyloid fibrils cause upregulation of presinilin 1 and cofilin 1. You could check this using RT-qPCR.
As mentioned above, your cells will represent certain aspects of the disease. You will have to check, which hallmarks of AD can be found in your model.
Good luck,
Sebastian
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Glutamate activates synaptic ionotropic NMDA receptors present in the post synaptic densities or PSDs, which sequentially activates the CAMK2 and CREB (cAMP-response element binding protein) that actively participates in neuronal plasticity, learning, and memory formation in adult brain, Then what is the exact role of amyloid beta 42 oligomers with respect to NMDA receptors a in causing dementia ?
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Synuclein Alpha and Amyloid Beta are two disordered proteins. A mathematical theory of AD progression was proposed by me (cf. preprint on RG). Memory reversal in this theory critically depends on guidance of Amyloid Beta, and two Synuclein proteins Alpha and Beta which compete with each other for surfaces available for binding.
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Thanks for attracting my attention to this review.
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How effective can i achieve my result when i have a "Labcycler 48" and a "NanoDrop" for Amyloid beta mRNA expression levels.
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Yes, you can study mRNA expression levels using conventional PCR, but… it depends on the type of study. If gene expression is low, then conventional PCR will not be reliable. The agarose gel can detect PCR products up to 10ng. Moreover, smaller the products, minimal is the binding with the dye (Ethidium bromide or SYBR green). So, sensitivity is compromised in conventional PCR. In other words, high copy number genes can be semi quantified using conventional PCR, but low copy number genes will be a problem.
For mRNA expression levels, real-time PCR is ideal which can either be qualitative (presence or absence of sequence) or quantitative (copy number). In RT-PCR, which would be more suitable in your case, the RNA transcripts are quantified by reverse transcribing them into cDNA first and qPCR is subsequently performed. QPCR can detect as low as 1ng and in real-time PCR, the accumulation of amplification product is measured as the reaction progresses, with product quantification after each cycle while in conventional PCR, the amplified DNA product, or amplicon, is detected in an end-point analysis.
Having a Nano Drop spectrophotometer is beneficial for both conventional PCR as well as Real Time PCR.
I hope this helps.
Best Wishes.
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Hi everyone,
I have gone through many papers of amyloid and phospholipid membrane interaction.
1. What is monomeric and oligomeric state of amyloid
doi:10.1016/S0006-291X(03)00386-3
Biophysical Journal Volume 83 November 2002 2610–2616
2. How to prepare monomer state of amyloid (25-35) and amyloid (1-42)?
3. What is membrane active amyloid or/and soluble amyloid?
DOI: 10.1038/srep20997
4. Is there any alternative of Trifluoroacetic acid for dilution or for keeping it in the monomeric state?
5. does the aggregation of amyloid occur even if low concentration of amyloid is used with time?
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Hi researchers!
We recently have bought amyloid beta 1-42 peptide from Genscript. According to the material data sheet, the peptide is soluble in water. However, after examining the amino acids, there are close to 45% of hydrophobic amino acids and 28% of charged amino acids.Theoretically speaking, this is not considered to be a hydrophilic peptide.
So, I was looking up methods already recommended by papers to dissolve this peptide. Some recommend dissolving in HFIP to remove the pre-aggregates and dry under a nitrogen stream. Following that, it is recommended to add a small amount of DMSO and the dilute with PBS or water. But some protocols mention that we could directly skip to adding DMSO. I only have 1.0mg of peptide and I am not to confident about adding HFIP and drying it as I might lose some peptides.
Thus, would it be fine to just dissolve in a small amount of DMSO and then dilute further with water? I do not think I can dilute the peptide sample in just water as it does not seem to be hydrophilic. If this method is fine, what would be the recommended volume of DMSO to be added to get a final concentration of 1mg/mL after adding water?
Thanks so much!
-Mathangi
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I agree with Andrei Blasko
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Its hard to detect amyloids even in brain tissue of Alzheimer patients using untarget proteomic methods. I am curious of what kind of protein is hard to detect by mass spectrometry, whether there are commonalities in their structures?
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Also hydrophobic regions (like transmembrane regions) which do not harbor charged residues (mainly positive charges, important both for fragmentation) are very hard to analyze with MS.
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I am looking to purchase the antibody components of the Wako ELISA kits for detecting Amyloid Beta 40 and 42 and am wondering if anyone else has done the same. Have you had good results with them, and what advice might you have regarding dilutions and protocol adjustments? I have optomized protocol for making the coating buffer, wash solution, reaction mixture, and stop solution. I will also be using TMB according to sandwich ELISA protocol. I am looking to purchase the antibodies separately because I need to conduct numerous ELISA assays and purchasing the pre-coated 96-well plate kits is cost-prohibitive. The antibodies I will purchase from WAKO are the coating antibody BNT77 (anti-human AB (11-28)), HRP-conjugated antibody BA27 (anti-human AB 1-40), and HRP-conjugated antibody BC05 (anti-human AB 35-43). I will be using amyloid beta peptides 1-40 and 1-42 purchased from rPeptide as diluted standards. Advice is appreciated! Thank you!
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I have not used the kit but WAKO is a trustable company purchased by Fuji. If you decide to use it, I would recommend doing a test run with your ELISA (positive and negative controls with it). The following mention ELISA which might be helpful (sorry it is not WAKO):
We also published a paper with beta-amyloid staining in C. elegans:
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I am trying to develop amyloid beta 1-42 i.c.v model in rats. Can anyone suggest after how many days of infusion can I subject the animals to Morris Water Maze to evaluate cognitive decline?
Also which administration, whether single dose, multiple dose, unilateral, bilateral administration is recommended.
Thank you in advance.
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Shin Murakami Thank you for your valuable response. I will check them.
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Hello,
We are trying to detect accurately the beta amyloid concentration of our solutions with ELISA kits, but we almost never get the concentration that we know that is been analyzed. We have tried different kits as we have read that some kits have BSA in its sample diluyent, but we still don't get it so we don't know what is the key. We prepare the beta amyloid solutions with HFIP and HEPES now, but we have already tried DMSO instead of HEPES. Anyone has any idea?
Thank you so much!
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Hi,
Just to begin with, make sure that your BSA is IgG free (i got one from jackkson immuno research). Else you will get erroneous results. Also the Elisa wash buffer should be filter sterilized. These are minor things but might help getting the accurate results.
Thanks,
Alpana
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Typical positive congo red spectral assay for binding to amyloids would be an observation of spectral shift from 498nm (CR only) to 540nm (in presence of amyloids). However I've tried the both the spectral assay and the birefringence assay (http://www.assay-protocol.com/biochemistry/protein-fibrils/the-congo-red-birefringence-assay) with an amyloid sequence AB(27-32) peptide that forms amyloid fibers, the spectra I've got does not shift to 540nm, there was only an increase in absorbance at 498nm. Under polarized light microscopy the fibers do appear to be apple green and birefringent after staining with Congo Red though. What is wrong? Or is it normal to get an increase in intensity of the spectra instead of spectral shift?
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Also had such problem. I use Congo Red for studying amyloids in a biofilm matrix. With comparable visualization of the staining density, I found that 540 nm works best for me, no more, no less.
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Dear researchers,
I currently prepare different protein aggregates (monomer, oligomer and protofibril) using methods in literatures and protect them by PICUP before running gels. Because previously I found my irradiation time might be too short, I elongated the irradiation time to 5s and 10s, but I still can't find any useful information after Coomassie colouration. There are no clear bands, only something like shadow. And there are no protofibril bands, whose molecular weight is over 100 kDa.
My gel percentage is 12%, and I use tris-glycine gel. The running voltage is 80V in 40 minutes and 120V in 60 minutes. I wanna know what could this gel tell me, and what I should do to see clear protein bands. Thanks a lot!
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Hi
Did you solve this problem?
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I am trying to form amyloid beta 1-42 oligomers for i.c.v injection in rats. As per WB. Stine protocol use of Hams F12 culture media is recommended but I am facing availability issues. Can I use DMEM instead of Hams F12 phenol free culture media?
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Thank you Iris Lindberg
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I could find only A11 antibodies which are polyclonal.
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The only one we know of at the moment is a recombinant rabbit mAb (M204) from Creative Biolabs: https://www.creativebiolabs.net/Anti-APP-Fab-Fragment-clone-M204-58679.htm
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I use HFIP for dissolving hIAPP (Amylin) fibrils to make monomers. I would like to ask Which solvent could be a replacement for HFIP to dissolve amyloid fibrils?
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Apart from HFIP, you can either use 2,2,2-trifluoroethanol (TFE) or dimethylsulfoxide (DMSO) for dissolving amyloid fibrils. DMSO is required in a higher amount (80%v/v). For further reading, you can go through the following articles.
1. DOI: 10.1007/s12551-018-0421-8
2. DOI: 10.1093/jb/mvg124
Best,
Avishek
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Hi everyone, I am a master student who currently working with a program detecting amyloid β aggregation, so I need to use Abeta 42. After treating with HFIP and get a peptide film, I want to dissolve it with 1% NH4OH. But after adding the solution and pipette up and down, there is much foam producing and can't disappear in several minutes. Have you met with this situation? And can I centrifuge for a few seconds? I am afraid it would aggregate if using centrifugation, because I need the monomer form. Thanks!
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Isam Eldin Hussein Elgailani Thanks Isam! I will read them^ ^
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Hi everyone,
I am a first year PhD student working on amyloidosis. I would like some tips and tricks to crack efficiently my current nightmare, i.e. the western blotting of the Amyloid beta and its oligomers. I am using brain endothelial cells to understand its presence in different oxidation condition, but as you might understand I have some problem with the WB technique (I am very naive about it).
I am using 16% tris-tricine gels, but my 2X Bio-Rad loading buffer contains Coomassie so, when i transferred my proteins to a 0.22 um NC membrane it stained horribly all the membrane, making impossible the detection of the amyloid-beta antibody with any detection method. Then another problem I got is that, since I need to load huge amount of proteins (around 60 ug), I had to overload my wells and the electrophoresis was horrible. All my bands were so thick and not sharp as they should be (or I am used to with tris-glycine gels).
So, do you have any advice on how to run effectively this kind of protocol? Also, any advice regarding how to obtain more concentrated lysate would be beneficial. At the moment my cell lysate has a concentration of around 2.80 ug/ul.
Ps: I am using a cerebral cortex human brain lysate as a positive control, together with synthetic AB40.
Thanks!
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Your protein concentration is not bad; I think it should work. Please follow the protocol exactly as described in the attached file. You may use the URL therein to watch the method video.
All the best!
Anoop
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Dear all,
I am currently working on amyloid beta aggregation. In ThT monitered aggregation assay, I have already obeserved an increase in ThT signal, indicating the aggregation of amyloid beta. However, after a certain time the fluorescence signal changes dramatically in all wells (as shown in figure). The concentration of A-beta I used in ThT assay is 5 uM and the concentration of ThT is 20 uM and the fluorescence signal was monitered for 48 hours. ThT emission was measured at 5min intervals with 3 sec orbital shaking before measurement. Besides, I sealed the 96-well plate before assay so I do not think it is the problem of evaporation. How can I optimized my experiment to get a smoother curve?
Thanks!
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Another possible contribution to the spikes is the formation of air bubbles in the wells.
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Hi every scientific explorers?!
Recently, I'm trying to study the aggregation process of amyloid-beta which is expression of its neurotoxicity and development of AD (Alzheimer's disease).
For now on, I know that the amyloid-beta becomes oligomer, protofibril and fibril as the aggregation progresses.
But there are some questions I want to know.
1-1. Does amyloid-beta monomer need to be misfolded (Conversion of α-helix to β-sheet) to become oligomer?
1-2. If it's right, what makes it happens? 1-3. And is it reversible or irreversible process?
Also, I read that amyloid fibrils were toxic. But these days, oligomers become toxic matters in aggregation.
2. Can you tell me the reasons why fibrils and oligomers are toxic in nervous system?
Thank you :)
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You raise very good questions, which are still open for discussion. There is no specific answer. We explore the processes involved in the different aggregation stages to eventually be able to definitely answer these questions. Amyloid aggregation is a generic process and you can find some answers in this review: . I hope it helps.
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This is our first time doing SDS-PAGE, and therefore we would like to hear from more experienced people if we interpret the results right.
Attached is a picture of one of our gels with our positive and negative control. These are from E.coli cells where on produce amyloids/curli (positive, P) and the other one is modified not to produce amyliods/curli (negative, N). Both controls have been through an isolation process to try and isolate only amyloids. Then some has been treated with fomic acid (P+ and N+) and some just with MilliQ water (P- and N-).
Here is our interpretation:
The formic acid-treated positive control, P +, shows a clear band between 14.4 and 18.4 kDa. This band is not seen in the sample with MilliQ water, P-, or the negative control. Thus, the band is expected to be amyloid monomers, as the molecular weight also corresponds to CsgA in curli, which is thought to have a molecular weight of 13-17 kDa. The thickness of the band may indicate a high protein concentration. In addition to this clear band in P +, several less clear bands are seen through the gel that can give an effect of smear. This suggests several different sizes of proteins have been present in the sample (not successful isolation). Also several clearer bands is seen for P + compared to P- (this we don't understand why).
In the formic acid-treated negative control, N +, several less clear bands are seen throughout the gel. This is due to proteins of many different sizes in the sample, as seen in the positive control. Also bands in N+ appear more clear compared to bands in N- (why?). In general, several identical bands are seen for both controls. This is to be expected as both controls are E.coli and therefore the same proteins are found in both controls except amyloid proteins in the negative control.
Generally there is a tendency of diffused bands, which can give a smear-like effect. This may be because the temperature during electrophoresis has been too high. A solution may be to run the electrophoresis for a longer time at lower voltage.
Please let us know if this is correct, if we missed something or other thoughts :-)
Thank you!
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The explanation given by you seems rational. However, to seek more information, perform MALDi TOF of thick prominent band.
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I am facing a difficulty in finding binding residues of beta amyloid. In articles, there are binding residues of helix of amyloid. Can anyone help me how to find active site and peripheral sites residues of beta amyloid.
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Ghulam Md Ashraf
I am looking for a docking study.
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The amyloid fibrils are aggregates formed by the self-assembly of monomers and it is generally dynamic phenomena that depend upon the nature of the analyte, solvent, and other solvothermal conditions. However, the monomers, oligomers and matured amyloid fibrils all present simultaneously in the solution, but they may affect the cell differently. So in this scenario, how we may quantify the amount of fibrils that actually responsible for their cytotoxicity.
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Hi Prabhjot,
Preparing samples for cytotoxicity assay could be a little tricky. For this purpose, you first want to separate out the different species. For example, if you want to study the fibrils for cytotoxicity, you either 1) use a cut-off filter to collect larger species/fibrils in the retentate fraction, or 2) carry out high speed centrifugation (ultracentrifugation, if required) and harvest the fibrils in the pellet. To use for a cytotoxicity assay, you will require to resuspend the pellet from ultracentrifugation and doing this might cause a little dissociation of the fibrils to release relatively smaller species; however the resuspended material is clearly a better option over a solution mixture of monomers, oligomers and fibrils. In case you are planning on studying the oligomers for cytotoxicity, you will either 1) need a special separation method like size exclusion chromatography or 2) samples collected at a very early stage of aggregation, when the fibrils are not formed yet. If you prefer the latter approach for oligomer collection, you should first follow the morphology of species formed in solution during the course of aggregation using Transmission Electron Microscopy or Atomic Force Microscopy (AFM), so that you will be sure of what material you are using for your cell toxicity assay. Having said that, none of the above described methods will help you precisely quantify what species you are dealing with, however these will definitely enrich one species over the others, and that eventually will increase the confidence level of what you are testing in a given experiment.
Hope this helps. Good luck!
Anoop
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I am trying to perform multiple immunolabelling for dystrophic neurites,amyloid beta plaques and Tau tangles for which I need antibodies from different host species. I already have mouse and rabbit isotype. I am looking for a third different host species. Any lead on this would be great help. Thank you !
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Sure, I will try this out
Antoni Iborra
. Thanks a lot for the info :)
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Hello,
I'm looking for information about what's the common size of Amyloid Beta clots, and When are they become dangerous? (i.e. is a 1nm diameter size for a clot is harmful and puts too much strain on the neurons?)
Thank you.
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Although the size of the amyloid deposits may play a role in amyloid pathophysiology, the functional aspect should be considered. Indeed, in the particular case of people suffering from amyloid deposits who are at risk of hemorrhage. It is likely that this risk may depend on the type of amyloid deposited.
indeed, discoveries in recent years highlight the role of a domain that has been shown to inhibit proteases, called the Kunitz protease inhibitor (or KPI) domain. It is present in several isoforms of β-amyloid (Aβ), it has the effect of inactivating certain blood proteins that cause coagulation.
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Hello,
From what I've understood, MRI works for imaging tissues mostly consisting from hydrogen. Since imaging of amyloid beta peptides is also available, I wanted to know what chemical element do Aβ mostly consists of? I wasn't able to find it online. I only found that for Aβ imaging, NMR was used, but from what I've understood it is used for analyzing many isotopes of different chemical elements, so it's not that informative. I would be very grateful if anyone could refer me to some reading material or refer me to a place where I can find this information.
Thank you very much for your time an attention.
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First of all, you seem to think that NMR and MRI are separate modalities, but actually MRI is a sub-discipline of NMR.
Yes, Beta Amyloid is a hydrocarbon just as all other macromolecules in the body. MRI in living tissues usually only sees the free water in the tissue, and depending on parameters, maybe the fat. The hydrogen atoms in macromolecules have shorter relaxation times and much lower concentrations that water, so they don't show up in typical MRI images. There are ways around these limitations, but distinguishing one macromolecule from another in the body, in an MRI experiment, is nearly impossible.
My understanding is that Beta Amyloid plaques can be seen in gradient-echo MRI images, because the plaques contain a build-up of iron, which leads to a very short T2* relaxation time. Therefore the plaques appear as signal voids in the images.
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I am treating neuroblastoma cells with AB42 protein (monomers and fibrils in separate experiments) and studying what happens after its uptake and concurrent interactions with intracellular exosomal machinery. Now, I plan to treat the cells for 24 hours, wash them with PBS and replace it with media without AB42 and incubating for another 48 hours.
In this time period I want to see if there is something happening to the AB42 inside the cell. For this I want to do a Western blot for the protein by preparing a cell lysate.
Are there any recommendations for a decent lysis buffer to use and whether or not I should use protease inhibitors? What are the best conditions for the Western Blot? I usually do it in a 4-12% Bis-Tris gel in 1X MES Buffer.
PS: I do not want to alter the structure of my protein for the western blot.
Thank you
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Hi Nandan
I wonder if you solved your problem and was possible for you to detect amyloid beta 42 by WB because I am currently trying to detect this protein with no success from an astrocytic lysate (cells transfected for APP). I know that the levels of amyloid beta can be really small, that´s why that I am trying to concentrate them.
Would be incredible helpful if you could give me any advice for the western blot protocol of this protein.
Thanks in advance
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I am trying to simulate AD conditions in rats using Amyloid Beta 1-42 oligomers for which I would like to know the best methods for its processing. I have read about the use of Hexafluoroisopropanol (HFIP) and Dimethyl Sulfoxide (DMSO). If any one has used these substances or any other better methods of Amyloid Beta 1-42 processing kindly let me know.
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I have never done it myself but have recently seen the poster at a meeting. In this publication, they are showing a method to generate the fibrils (1 week at 37°C shaking at 100rmp). Also, they describe that the fibrils form the brain differ from the ones made in vitro.
Best,
Oliver
Kollmer, M., Close, W., Funk, L. et al. Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue. Nat Commun 10, 4760 (2019). https://doi.org/10.1038/s41467-019-12683-8
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We are expressing B-amyloid peptids and after purification we are having some problems with the aggregation of the samples. I'm wondering if there are optimal conditions of storage that dont include liophilization of the sample.
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Lyophilization of the peptide is the best way for storage. When lyophilization is not available, the purified fractions may be may flash-frozen and stored at -80 °C; however, the peptides should be run through a gel filtration column before you perform the assay.
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Amyloid beta 1-42 in PDB is 1IYT is used for docking studies. similarly what is the PDB id that is used to dock insulin fibril with the liagnd for docking studies.
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Thank you Kapil Mehta for your reply
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I am trying to do a kinetics characterisation of AB-40 and AB-42 (they are recombinant peptides produced in the laboratory) but it seems that the samples ar alredy aggregated when I try to do the kinetics mesurement. This peptids, after the purification procedure, are storaged at -20°C and in a solution with a pH of at least 8. I was trying to disaggregate using sonication but it doen't work so well, i was wondering if there is another method I can use for the sample preparation.
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You refer to amyloid beta peptides that are highly hydrophobic peptides. I came across the following paper that might be useful to you:
Broersen, K., Jonckheere, W., Rozenski, J., Vandersteen, A., Pauwels, K., Pastore, A., ... & Schymkowitz, J. (2011). A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease. Protein Engineering, Design & Selection, 24(9), 743-750.
If you have a powder form of the peptide you could ‘simply’ use DMSO or TFE (after TFA treatment) see for example:
Killian, J. A., Keller, R. C. A., Struyve, M., De Kroon, A. I. P. M., Tommassen, J., & De Kruijff, B. (1990). Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes. Biochemistry, 29(35), 8131-8137.
Where a highly hydrophobic signal peptide was used.
Hope this helps you a bit further.
Best regards.
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I am currently working on a system that contains a DMPG bilayer with amyloid beta monomer on one side of the membrane. I generated the system using CHARMM-GUI and oriented the protein using the OPM plug-in. I choose 1:1 as the ratio of DMPG in the upper to lower leaflet. However, after equilibration in GROMACS, I used GridMAT-MD to look at the area per lipid, and saw that I had one leaflet at ~62 sq A and the other at ~57 sq A. The literature value I found for DMPG is 62 sq A per lipid I'm guessing that the protein made one of the layers more tightly packed than the other. How do I adjust the CHARMM-GUI settings to make sure that the area per lipid is even on both sides? Or is there a way to 'fix' the system as it is?
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Wojciech Kopec
that makes sense. The protein is asymmetric (resting along headgroups of one leaflet) and the paper I was looking at was for a pure lipid membrane:
I had difficulty finding literature on area per lipid in protein-membrane systems, which is why I was concerned. Thank you for the clarification.
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I have tissue that have been stained with Thioflavin-S as wll as an HRP immunostain for beta amyloid plaques in Alzheimer's mice. I mainly want to quantify the plaques by the amyloid load (the percent area it takes up in a given region) using imageJ but I haven't really used the software before. Any ideas on how I can do this? Some video tutorials would be greatly appreciated.
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The materials and methods section of our paper "Hippocampal administration of chondroitinase ABC increases plaque-adjacent synaptic marker and diminishes amyloid burden in aged APPswe/PS1dE9 mice" may be of use to you as it gives an overview of our use of Image J to quantify plaques by amyloid load in an AD mouse model. The full text is available on my profile page.
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It is well known that most of the pathological protein deposits contain several different components/proteins, along with the main protein constituent. What are the proteins or components that commonly found in ATTR deposits?
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Dear Behnam,
Thanks for these references, I should have a close look at these.
Best regards,
Mahafuzur
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Im working with a His-Tagged protein that aggregates (amyloid like) on nervous system neurons and I need to evaluate the kinetics of this process. Do I need to remove the His-Tag of the protein?
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The answer is in the question. You are evaluating the kinetics of the process of aggregation for your native protein, not the his-tagged one. To be sure about the influence of the His-tag, you need to compare it to the untagged protein. Remember that most His-tag contructs that can be cleaved off by a protease leave additional amino acids on you protein which can also have an influence.
So the best option would be a construct where you can get the native N-terminus free of additional amino acids. Purifying untagged protein is more difficult, so you could use e.g. the His-SUMO fusion tag which can be cleaved by its protease.
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I have purified a recombinant M0 AB(1-42) using a recent method from
I have used 15mM NaOH as SEC buffer and also tried to keep it in monomeric state by bath sonicating for 1 minute. I have also tried filtration of these peptides using 10 kDa cut off centricon. I put my peptides for aggregation after adding it in pH 7.0 sodium phosphate buffer and maintaining the pH of the initial peptide solution to 7.0 and initial concentration to 50 micromolar. I find out concentration of abeta using absorbance at 293 nm and extinction coefficient 2400 mol L-1 cm-1 . I set the amyloid peptide solution at 37 degree Celsius and 200 rpm in orbital shaker incubator. Still the solution is not showing any turbidity and also no increase in thioflavin-T fluorescence even after 72 hours. I also obtained circular dichroism spectra and I am getting only random coil in my peptide solution as implied by the spectra even after 48 hours. I just observe thread like entities floating in the solution but no increase in turbidity. Please help me out with this problem. I suspect amorphous aggregate formation.
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Dear Vanessa,
I have tried everything possible Still i can't get any aggregation. I checked by EM and it seems to form amorphous aggregates. What could be the reason for non aggregation into amyloid fibrils for such a high propensity amyloidogenic peptide. I am in desperate need of help. Please help me out.
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I want to collect exosomes from SHSY5Y cells after treatment with 1µM Amyloid beta and then measure the Amyloid beta concentration of the exosomes. I'm not sure if the concentration I'm treating the cells with is sufficient for detecting Amyloid beta in the exosomes, or if I need to increase it.
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If you use flow cytometry and immunofluorescence labeling, then there are different concentration reported to see the expedient results in different cells.
However, I have seen a few reports using 1 um Abeta through exosomal labeling, and they all got sufficient amount of Abeta to be detected in exosomes.
Example:
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I am trying to develop an efficient and an effective AD model using Wistar rats.I require suggestions as to which model may be the best one to mimic AD pathology in rats. The one that comes to my mind is Amyloid Beta 1-42 i.c.v injection. But a wide range of Amyloid Beta 1-42 are available so would like to seek advice from the fellow researchers who might have used it in their work. Also would like to know if any other better routes of administration are available or not, as i.c.v is an invasive one.
Note: I am looking out for non- transgenic models of AD.
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I also agree with Bahman Sadeghi , that your hypothesis can influence the choice of the model.
However, since you are looking for non-invasive and non-transgenic models, I would suggest using Aluminum chloride (AlCl3) or D-galactose models. Various studies have used these models to mimic AD-neurotoxicity. Some useful Links:
AlCl3:
D-galactose:
I hope this helps, Good Luck.
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Flutemetamol and Pittsburgh Compound B are used as Amyloid Binders used for Alzheimer's disease diagnosis.
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Just still a few additions: EP2182988 is still an older granted patent and WO2007064773 (University of Pittsburgh), which is withdrawn in the European phase. So, considering all these data and knowing that the University of Pittsburgh applications are not valid in Europe; an infringer or counterfeiter can only be accused by a patent proprietor with a valid patent in those countries where the patent is still valid. In the particular case of Markush claims (=many substituents on a basic structure), invalidation of a patent can be supported by showing that replacing one substituent by another has no 'inventive step' and can be considered as obvious for a skilled person. By the way, this short analysis is only for Europe.
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Can anyone help me with a working protocol for amyloid beta detection by western blot? I have extracted the whole proteins from the cells. For the Western blot I used a Precast gel 4-20% (in SDS condition) and then for the transfer I used the nitrocellulose membrane. For the immunoblotting I used anti-amyloid beta42, clone G2-11(Millipore) but it doesn't work. Anyone have some advices or protocol for me? Thank you!
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Yea, if the control isnt working then that is another issue. It may be that you are running the gel too long and its basically being pulled through and out so you get negatives throughout. I would also try to have an beta-amyloid control if possible too.
I have my old BSc thesis uploaded here and looked at something similar, may be of some help, may not. I was looking at more specific regions of APP rather than amyloid itself
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Hi,
I have a filamentous protein which has high beta sheets content and tends to aggregate. I speculate that such aggregation was induced by the beta sheets similar to amyloid but I don't want to refold the protein and prevent beta sheets formation since it preserves the function of the protein. I am wondering what will be a good way to deaggregate this protein? I have tried some common detergent (such as OG and Tritton) or amphipols, but it barely worked. Amphipols seemed to make the protein soluble, but did not solve the aggregation issue.
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Manuele Martinelli Thank you for your answer. When I ran gel, I always add the reducing agent. Maybe I should try and compare how differently it will show. Meanwhile, there is only one cysteine in my sequence, but I am not sure if that's the reason of aggregation. Maybe I can add some reducing agent to see if it solves the aggregation problem.
Best,
Yangqi
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Hey,
We are looking to study amyloid beta (1-42) and prion protein in a co-culture and separate culture system on neurons and astrocytes.
There seem to be a lot of different amyloid beta preps out there, I was wondering if anyone had an opinion or has had success with a certain companies preparation.
Thanks
Igal
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You can have a look at one of our recent paper for the preparation of amyloid stock and let me know for any concern.
Good Luck!!
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Shift of ThT excitation maximum (from 385 nm to 450 nm) and its emission maximum (from 445 nm to 482 nm) occurs only upon binding to amyloid fibrils ( doi:10.1016/j.bbapap.2010.04.001). And we do not see the formation of amyloid fibirls in the lag phase, right? Then why does the shift occurs at such early "lag phase" stage?
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Devanshu Mehta - Amyloid fibers are likely bound to the surface of the cuvette from a previous experiment. SDS is often not sufficient to remove them. Try washing in aqua regia
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I came across some protocols using Formic Acid in the first step of Amyloid beta extraction from cultured cells.
Formic Acid is not the safest substance to use, so I was wondering if there is a proven safer protocol that can be used to extract Amyloid Beta (both soluble and insoluble) from cultured cells.
I am interested in extracting it from Astrocytes.
Thanks for any advice.
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Yes! Most of the protocols use formic acid as a component at the purification stage like the following famous one:
But check the following older papers. Maybe you can find some useful methods without that stage:
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Hi
Dear all
I used aggregated amyloid beta 1-42 (10 micromolar) to induce Alzheimer's condition in PC12 cell line, but after 24 h incubation, MTT assay was done, I obtained 85% cell viability, I have a question and I would like to guide me?
is it possible to achieve the alterations such as hyperphophorylated proteins using molecular analysis like western blotting or RT-PCR while we did not observe cell death (50%) using MTT assay?
I'm looking forward to getting your answer,
Thanks a lot,
Bets wishes
Marzieh
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In general - and if results of otheres (50% reduced viability") are reliable - WB will not be an adequate compensation for mising viability data. However, you could perform alternative viablility assays, such as Calcein AM assay, or poptosis testing (AVPI staining), or analyses og Bcl-2 vs BAX expression etc. to determine viability, apoptosis and/or surcival of cells. This depends on the functions you want to determine, such as cell proliferation (MTT or CFSE staining), cell viability (calcein AM assay), apoptosis/survival (expression Bcl2/BAX or better AVPI assay (FACS method)).
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Hi, I am working with amyloid protein. For my experiment, I used lipid vesicles in the reaction mixture. After reaction is completed (amyloid formation), I want to isolate these fibrils from lipid vesicles for mass spectrometry analysis.
Can any one suggest me for the best method for isolation? Your suggestions will be greatly appreciated!
Many Thanks.
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Wash with chloroform. Lipids should be totally dissolved.
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I know extracellular amyloid deposition begins around 2 months in the transgenic mice, first in the subiculum and layer V of the cortex. Also, Plaques are found throughout the hippocampus and cortex in six months.
However, I wonder which part is more faster between hippocampus and cortex?? If something is not the first, is it happening at the same time??
Additionally, how about the human brain?
Thanks a lot! 
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In general, and in mice model of FAD (such as 5XFAD) the expeditious emergence and development of A beta plaque deposition has been shown to be at hippocampal pyramidal and granular neurons in the CA1 and DG.
Some data has interestingly shown that even microglia derived from 5 week old 5XFAD mice can impede A beta plaque formation in vitro:
Even though it would seem to be rather counterintuitive that cortical neurons can develop and deposit A beta plaques and NFTs earlier and faster than hippocampal neurons, there is not sufficient data to prove such rivalry! IHC and blotting data also have so far focused on hippocampal neurons as the cells that exhibit such deposition first.
It can also be inferred that the speed of AD-like symptom emergence and cognitive dysfunction is relatively higher in hippocampus compared to cortex in particular in primates and humans. I think that this can simply be explicated with regard to the smaller size and less neuronal density of hippocampus vs the cortical layers.
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I am studying the glycation mediated amyloid fibril formation. When there is native protein + fibrils i am able to see the bands in Tris-Tricine SDS PAGE. But, when i precipitate the fibrils, by centrifuge (12000 RPM, 30 mins), i was not able to see the band of fibrils. I tried even native page. What could be the reason? Many thanks for your suggestions.
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Electrophoresis of SDS-resistant amyloid proteins is described for example in doi:10.1016/S0076-6879(06)12003-0 and a video on
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Test group
-5xFAD female 4 month-age Tg (n=8)
-5xFAD female 4 month-age Non-Tg (littermate of Tg) (n=7)
(Amyloid beta is only generated in Tg mouse, not Non-Tg)
Behavior test
-Morris Water maze
- Contextual fear conditioning test
Result
-In both tests, Non-Tg mouse behavior is worse than Tg.
Questions
-What could affect these unexpected result? I expect that Non-Tg Behavior is better than Tg.
-I suspect that their father and mother age were old when mating (5 month-age).
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Depening on what your actually readout is, 4-month-old 5xFAD TG mice do not show any behavioral phenotype compared to non-TG littermates at that age.
I could not even detect differences in older (7-month-old) mice. We usually dectect first behavioral differecnes in 10-12 month old females in the Morris water maze, but this is highly dependent of the setup and experimental procedure.
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Hi everyone,
I am trying to detect Amyloid oligomers in brain tissue extract of APP mice by ELISA kit but the outcome is mostly undetectable.
I am using IBL-Elisa kit: Amyloid Beta (82E1-specific) Aβ Oligomers (ref27725) and I have problems detecting it. I homogenize 5-10mg of tissue in Tris buffer pH7.4 (20mM Tris; 140mM NaCl) with a pestle (not sonication).
I have searched for publications about this form of samples processing but I don’t get any clear suggestion about it.
Anyone knows if it is necessary some other protocol or previous-step for low-weight samples?
Any advice are welcome.
Thanks in advance!
Ángel
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Hi Santosh Jadhav , I have checked the link that you pointed me out but I need to assess low-molecular weight oligomers that coexisted with Abeta plaques and soluble Abeta40 and 42.
Thanks!
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Dear all,
I need to quantitatively assess Aβ oligomers in transgenic mice (5xFAD) of 6-8 months as well as 3-4 months and 10-12 months.
I was thinking of using  IBL-Elisa kit: Amyloid Beta (82E1-specific ) Aβ Oligomers ref27725. 
However, my samples are frozen brain tissue and I don’t know how to prepare them. Can anyone share his experience in preparing the samples for such analysis?
IBL gave me two protocols :
1-  with CHAPS buffer
2- with Guanidine
The issue is that they were not able to tell me wether any of the protocols were suitable to this kind of analysis with their kit.
Moreover,  does anyone know if neat Aβ oligomers can be detected in 6-8 weeks old mice ?
Any recommandation about other detection kits are warmed welcomed!
Thanks in advance.
Best regards,
Eva
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Hello Eva Ploux ,
I carry out this type of experiments actually. I had been doing a few probes with tissue of transgenic mice of 26 weeks of age and as result, I have obtained very low values or mostly undetectable…. I use tris buffer Ph7.4 (20mM Tris, 140mM NaCl) with phosphatase/protease cocktail inhibitor for homogenized without sonication.
I use the same ELISA kit that you have mentioned in your post. Have you had good results with these Kit?
Thanks
Best!
Ángel
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Hello People!
I have been thinking about using designer macrocyclic peptides to hydrolyze the backbone of the amyloid peptide (in the aggregated form). Tentatively - the cysteine protease mechanism. It's a long shot - catalytic peptides are a new frontier, yet this is what could be a game changer for advanced stages of Alzheimer's. Anybody interested in a collaboration? Greg
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In general, it is far better to prevent things from happening in the first place than to wipe the blood from the floor afterwards. This is particularly true for neuronal diseases, even if the regenerative ability of the brain may be larger than I was taught it to be when I was a student.
And another thought: soluble precursors of amyloid plaques and tangles have a much larger chance to interact with something important (and break it) than the insoluble precipitates. If I had to bet, I'd put my stake on them, just like Alzheimer did in 1911 (English translation in doi:10.1177/0957154X9100200505).
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Dear all,
We work with an animal model of Alzheimer's disease, and would like to quantitatively assess the presence of Abeta oligomers (ADDL or lower species, if possible) in different brain regions.
Have you ever successfully used an ELISA kit for this purpose? If so, would you mind sharing your experience (with a publication link, or product number and company)? While the WB technique doesn't have our preference, I would also appreciate any feedback.
Thanks in advance.
Best regards,
David
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We published work while at Washington University in St. Louis using an N-terminal specific monoclonal for both capture and detection (similar to 82E1). There is also information regarding sample preparation in both papers.
In my view the quantitation with such an assay is only giving you part of the picture. You will miss oligomeric components with N-terminal modifications or truncations.
We have moved our lab to the NIH and have pivoted our focus yet remain actively interested in the topic. Feel free to reach out with questions or thoughts.
Best,
Thomas
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I have been setting up aggregation reaction for B2m since a long time. However, I have not been able to obtain pure (any) fibrils of B2m till now. I am briefing the condition that I have checked and its result.
Firstly about the B2m-
a. The protein has been recombinantly expressed using BL21DE3 cells.
--The protein has a methionine at the N-terminal.
b. The purity of the protein was assessed using MALDI-MS.
c. The secondary and tertiary structure has been assessed using CD Spectroscopy and Fluorescence spectroscopy, respectively.
d. The presence of di-sulfide bond in the purified protein has been assessed employing native and non-native conditions in SDS-PAGE.
Aggregation conditions for B2m-
Since my work involves experiments near-physiological conditions, I have worked to generate amyloids using copper ions. (No additives like SDS, TFE are used to keep the conditions near-physiological)
a. I have used (as per the reported literature) 100 uM of B2m, 200 uM of CuSO4, 200 mM of Potassium acetate, 25 mM MOPS, 500 mM Urea and incubated the reaction at 37 deg C without agitation. However, I have not been able to get any amyloids. There is no increase in ThT intensity with days. I have checked it at 10th day and 20th day of the experiment using TEM. I have attached few TEM pictures for consideration.
b. I am now working with acid conditions to generate amyloids of B2m and use their seeds at physiological pH.
1. Can anyone help me to understand where things are going wrong?
2. If there is any particular conditions that might be set?
3. I have observed increased ThT intensity once in my initial try to generate fibrils using the same conditions as explained above, later confirmed using AFM (pic attached). However, it seemed a heterogeneous population of aggregates then. But amyloids did form. However, I have not been able to replicate the experiment with then obtained results.
4. The N-terminal being involved in copper binding is what I feel might be an issue. After setting the aggregation reaction at above conditions now I always get precipitate in the control (No copper, but 10 mM EDTA) and the reaction tube within 10 minutes (which was not the case during my initial try).
*I have tried replicating the initial conditions for generating amyloids. I purify protein using PBS pH 7.4, which later, I buffer exchange into 25 mM MOPS pH 7.4 using SEC before setting the aggregation reactions using the above condition.
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Hi, Thanks for the reply.
Yes, the concentration of CuSo4 that has been used in prior optimization of setting aggregation reaction (as per already available literature) isn't physiological and the concentration of B2m being used isn't as well. It is the ratio 1:2 (B2m:Cu(2+)) which has been observed as important.
Can with time the amorphous-like aggregates turn into mature amyloid fibrils? I have observed the aggregation reaction till 20th day (which is more than 2 weeks, as per the provided information in the literature). However, I will look into it and wait for some more days.
"... push the molecules a little harder..." I had tried PBS, Tris and MOPS buffer, at different concentration of proteins (10, 20, 30 ,....100 uM B2m). In all the set reactions 'good amount of precipitation' was common, clearly visible within 10 minutes of setting the reaction. And ThT binding assay performed for 10 days of incubation showed no increase in intensity.
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i am looking forward to increase amyloid precursor protein in my SH-SY5Y cell line. Can anyone share their experience and protocol.
Thank you
Parth Naik
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Hello, our Viromer RED transfects quite good shRNA or DNA plasmids into SH-SY5Y cells...
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I currently have amyloid beta 1-40 peptide at a concentration of 80uM in 40mM Tris Buffer at pH 7.5. The peptide comes directly off a size exclusion column. I need to run my reaction at 500uM amyloid beta 1-40 in an acidic solution (pH<6.5) of sodium acetate buffer. How should I proceed?
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Ouch, from my experience this can be very difficult, I would suggest to dialyze it against water, lyophilize (dry-freeze) it and then add the volume of buffer needed to perform your reaction at the desired concentration. In order to prevent the formation of aggregates from the resuspension of the lyophilized protein, you can thaw it in NaOH 3.5 mM and then add an equivalent volume of your buffer 2x concentrated (with the sum of the two volumes being the one that make you reach 500 uM of peptide concentration). The high pH prevent the protein from aggregating instantly.
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I plan to seed neuroblastoma cells. Differentiate them with retinoic acid, treat them with AB1-42 and then stain them with Congo red to observe amyloid aggregates in those cells.
Thank you in advanced
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If you - what we do not know for now - process the mentioned neuroblastoma cells from culture to embeddings (frozen or cryo-sections or Paraffin-sections) and have access to ResearchGate you might enter the following URL (when you already are in ResearchGate website):
where you'll find some Staining recipes... too
e.g. [001] Jakov Milić Re = now 2 year ago [=Mar 10, 2017]
&&
Best of luck,
WMH.
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I need a suggestion on the techniques I can use to study the formation of amyloid from lysozyme enzyme and also the effect and inhibitory mechanism of small molecules in it's formation.
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Hi Debanjan,
you can use Thioflavin T (ThT) dye, it is an amyloid specific probe, using fluorescence spectrophotometer or plate reader you can in real time follow the amyloid aggregation kinetics. Increased ThT intensity you will observe if lysozyme is forming amyloid type of aggregation and in the case of inhibition study decreased ThT intensity compared to control you should observe. you can follow my article for various biophysical tehniques to follow kinetics of both aggregation and inhibition.
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Hi, everyone,
I would like to quantify the Abeta load of my IHC staining images. How should I do it? someone use plaque area %, someone use plaque numbers, which one is better? And can I do it with Image J or Fiji?
Thanks so much!
JInjing Yao
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You should be able to quantify it. Do you have an example image?
- Melissa
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Drug from Pfizer, design from Pfizer, researchers from Pfizer, statisticians from Pfizer and the result: drug works well!
It is not necessary to read whole manuscript!
"Maurer MS, Schwartz JH, Gundapaneni B, Elliott PM, Merlini G, Waddington-Cruz M, Kristen AV, Grogan M, Witteles R, Damy T, Drachman BM, Shah SJ, Hanna M, JudgeDP, Barsdorf AI, Huber P, Patterson TA, Riley S, Schumacher J, Stewart M, Sultan MB, Rapezzi C; ATTR-ACT Study Investigators. Tafamidis Treatment for Patients with Transthyretin Amyloid Cardiomyopathy. N Engl J Med. 2018 Sep 13;379(11):1007-1016. doi: 10.1056/NEJMoa1805689."
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Dear Mesut
I have started to believe that there is politics to reach.
Journals define best articles, best articles get featured and attract systematic reviews and attract citations, get published in these journals and raise their impact (making them best). A phenomenon of you scratch my back, i scratch yours.
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As we know so far, around 25 proteins and peptides are known to involve in amyloid disease. If we assume that the fibrillation is an intrinsic feature of all polypeptides, and that we have tens thousand of proteins and peptides in body, why only a few of them convert to toxic aggregates and go to fibrillation process?
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Proc Natl Acad Sci U S A. 2002 Mar 5; 99(5): 2754–2759. doi: 10.1073/pnas.052706099PMCID: PMC122420PMID: 11880627Biophysics
Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation
The above is quite informative, although it may not necessarily paint the whole picture.
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I came across a paper exploring the binding kinetics of thioflavin T and amyloid beta fibrils. The fibrils were monomerized initially and prepared dissolved in CAPS-guanidine (CG) buffer. Does anyone have an idea as to the functional purpose of this buffer? Please include references if you have any. Thanks!
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  • CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulphonic acid) is a zwitterionic detergent that helps to dissolve hydrophobic substances.
  • Guanidine (usually in the form of its hydrochloride GdHCl) is a caotrope, an agent that interferes with protein-protein interactions both between different proteins and within a single protein (leading to unfolding).
Both substances are routinely used to dissolve aggregates. You should read a good book on biochemical laboratory methods, of course I am partial to my own (ISBN 978-1-4419-7250-7)
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I am having difficulties to image protein fibrils using AFM. What is the ideal concentration of amyloid fibrils that I should use? Also how long does it need to sit on the mica surface before I dry it off?
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I'm quoting this from my labmate's paper:
Role of Small Oligomers on the Amyloidogenic Aggregation Free-Energy Landscape
doi:10.1016/j.jmb.2009.10.019
"Each day, after inversion of the tube once, 20 μl of the incubated solution was aliquoted for AFM imaging on modified mica. To modify the mica surface, we applied 20 μl of 0.1% (vol/vol) APTES (aminopropyltetratheoxysilane, catalog no. 151081000; Acros) evenly on a freshly cleaved 9.9-mm-diameter muscovite mica disk (product no. 50; Ted Pella) and allowed it to react for 10 min. Unreacted APTES was rinsed away with 15 ml of Millipore water. The surface was dried with HPLC-grade compressed nitrogen gas. The incubated sample was applied evenly on this freshly prepared surface and allowed to adsorb for 3 min. Unbound species were rinsed away with Millipore water. Residual water was blown away with nitrogen gas. The sample was imaged by a MultiMode Scanning Probe Microscope with a Nanoscope IIIa controller (Veeco), with a tapping-mode etched silicon probe (TESP; Veeco) in tapping mode in air. The scan speed was 1 Hz, with an image size of 512 × 512 pixels. Samples were stored in disk carriers when further imaging was required."
best of luck!
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I will be treating SHSY5Y cells with amyloid beta 42 obtained from E. Coli cultures. I observed that after treatment, the media became cloudy within 24 hours indicating a contamination. I did not pass it through a sterile filter since the protein may attach to the membrane and thus reduce the working concentration required.
Is there any other sterilisation method known that will prevent contamination of the cell culture media and also minimise the loss of protein for treatment?
Thanks!!
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Can you add ethanol to your amyloid samples and remove it after a while? you can use a vacuum centrifuge for that. Amyloids are very stable, so maybe ethanol treatment is not affecting them in any way.
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We are working on a new compound for Alzheimer disease and use THP-1 cell line and Amyloid beta 1-42 from US peptide as stimulator. Since, US peptide does not provide this Amyloid beta anymore we have to buy Amyloid beta from another company but I am not sure can I peruse the rest of my experiments using another Amyloid beta or I should redo all of my experiments including PCR using the new one. Either way, does anyone have any idea which Amyloid beta 1-42 normally is being used for this disease and cell line?
Special thanks in advance for your kind help
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Dear Davoud,
As I have seen in the literature, AB-42 is the most abundant amyloid beta in diffuse plaques.
Of note, ketamine and xylazine are also being used in order to develop AD in animals.
For the case of brand change, In my opinion, you can make a comparison in two different brand treated groups and then decide to redo your experiments or not.
Best,
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I want to check cell internalization of Abeta (1-42) peptide w.r.t. However, I do not have fluorescent labeled Abeta peptide to perform this experiment. Is there any other ways by which I can monitor the same.
Or is there any protocol to tag fluorescent dye to a peptide after cleaving it from the resin???
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Hi Jahnu, you could always do the classical internalization assay using radiolabeled I125, C14 or H3 ligand.
FlowCy and confocal are subjective for quantitatively determining internalization. Using radiolabeled ligands gives you an absolute quantitative value.
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