Almost all of the amyloid fluorophores I know of are in this file

I'm quite new in the field of amyloid beta and working since a few months on an anti-aggregation assay. I would be really interested in your project of shared protocols. I'm facing problems with batch to batch reproducibility: would you have recommendation about suppliers and storage? (I'm using Abeta1-42 from Anaspec, resuspend in HFIP, aliquoted, evaporated with vaportrap and stored with N2 gas at -80ºC).

There are two main sources of batch to batch variability.

The first is the concentration. Although the product is listed as 95% pure that refers to the protein content only. The actual sample is 30-50% salt, so the 1 mg you get from Anaspec is actually only 0.7-0.5 mg. This value also changes over time and is different for every manufacturer. Anaspec lists it at the bottom of the paper that came with the peptide under "Peptide Content".

For other manufacturers, you will need the actual concentration. You will also need the actual concentration if you do filtration steps. This is easy to do by reading the concentration at 280 nm. The absorbance values

This protocol gives a largely monomeric, very slowly aggregating solution (based on procedure of Teplow) : 1. dissolve peptide in 1% ammonium hydroxide, aliquot, and lyophilize 2. dissolve lyophilized peptide in 1 mM NaOH at 4C 3. bring to pH 7.3 with 10x concentrated zinc free buffer 4. Filter through .2 um filter This procedure will give you almost completely monomeric peptide at the beginning if the concentration is less than 200 uM. Be aware that aggregation from monomer is really, really slow (time scale of weeks), however, it speeds up considerably if stirring or shaking is used.

The reference for the original procedure is Methods Enzymol. 2006;413:20-33.

The modified procedure (Suzuki Y, Brender JR et al , Biochemistry in press) has an two step solubilization steps in NaOH and is performed at 4C. In our experience this enhances the relative fraction of monomeric vs. aggregated peptide. Using a size exclusion column instead of a filter is better if you have easy access

(Response from Laure Grand Moursel)

I'm indeed dissolving my aliquots in DMSO but would like to test different protocols without. I suspect DMSO to interfere with my assay.

What kind of filter do you use, any specification? Don't you loose a lot of protein during that step?Did you ever try to dissolve Amyloid beta in urea 8M? And then decreasing the concentration to have different amyloid species?

We usually use 0.2 um Spin-X filters, although 100 K or 50K filters are also a good choice. You lose about 30% of the protein.

Dilution from urea has been performed, though it less extensively characterized. The starting point from 8M Urea pH10 to 0.4 M pH 7.3 is monomer with 5-7 nm rod like aggregates (around 37%). Note that the initial pH is important. Depending on the assay this may be good choice.

And also this reference

(From Srikant Sarangi)

I had a quick question regarding the protocol you had suggested. Is the lyophilization important after dissolving in ammonium hydroxide if the sample is going to be used immediately.

In response to your question, the lyophilization is important if ammonium hydroxide is being used (1% =550 mM which will have large salt effects on the aggregation). If you don't want to or can't lyophilize you can try 1 mM NaOH instead of NH4OH, although the amount of aggregated material in our experience is somewhat more. It is difficult to store non-lyophillized samples (freezing and thawing can cause aggregation)

If your assay tolerates it, you could try dilution from DMSO or urea instead

Started to add papers to the project, particularly regarding experimental artifacts that are common with amyloids . I am particularly interested in protocols for the solubilization of ABeta to generate different oligomers and peoples personal experience with these. Feel free to add whatever papers, protocols or other documents you feel are appropriate.Especially looking for someone with cell-culture and in vivo experience to contribute.

An extensive list of amyloid fluorophores has been added by Steingrimur Stefansson

Hi, the following paper by Broersen and colleagues nicely describes a detailed and robust protocol to solubilize the Abeta peptides with very reproducible aggregation/toxicity behaviour in biophysical and cell-biological assays. I have excellent reproducibility for Abeta peptides with this methodology.

Broersen K, Jonckheere W, Rozenski J, Vandersteen A, Pauwels K, Pastore A, Rousseau F, Schymkowitz J.

A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease.

Protein Eng Des Sel. (2011) 24(9):743-50

http://www.ncbi.nlm.nih.gov/pubmed/21565831

thank you Jeffrey, summarizing various groups experience could be quite useful.

Let me share some of my own experience

1. Oxidation of methionine could be a source of irreproducible aggregation. Generally oxidized peptides tend to be more soluble. Lyophlized powder storage in the freezer allows to avoid this. Of course one can always use mass spec to make sure everything is all right.

2. As Jeffrey mentioned above, some salt may still present in the lyophilized material when you buy it from a company. Another thing to keep in mind - you do not actually know how they purify the protein and handle it afterwards. If it sits for some time in an aqueous solution before being lyophlized or something, well, then it may start forming fibrils.

3. people use different methods for improving reproducibility. We usually dissolve Abeta in DMSO at high concentration (~5-10 mM), but do not allow it to sit for a long time on the bench - it forms some sort of aggregates in the way. At low concentrations, though, DMSO tends to dissolve fibrils.

When DMSO interferes for some reason with other stuff, HFIP pretreatment could be used. There are two ways to go afterward: dry under a stream of nitrogen until a transparent film of peptide is formed as mentioned by Laure, or just lyophlize. I like the latter more, because dissolving the film afterwards is a pain.

The other approach which I tried when DMSO should be avoided, is dissolving Abeta (~100 uM) in 20 mM NaOH (pH ~12 mM) then titrate pH to 7.4.

Thanks to Kris and Alexey for sharing. I'll copy these comments to the main thread on the amyloid project bench. Regarding both comments, the best method depends a lot on the particular assay. A few comments based on Prof. Potapov comments and our own experiences.

1. Oxidixed Abeta also gives a distinct NMR signal which can be used for detection. I haven't seen oxidation of methionine in amyloid beta in real time NMR over a period of days in phosphate buffer in the absence of DMSO. We haven't looked in the presence of DMSO or over a longer period (Our recent real-time NMR paper on Abeta over a 90 day incubation period uses a 19F substituted methionine which will not oxidize.)

2. Depending on the source it can be a lot of salt (up to 60% of the listed weight for recent IAPP lots from Anaspec). Anaspec lists the salt content. I am not aware of others that do.

3. Also be aware that Abeta(1-42) (but not Abeta(1-40)) from Genscript are actually click peptides that are converted to the native peptide at pH 7.4. If this particular peptide is used at a lower pH it may not convert from the unnatural O-acyl form to the native peptide

4. Personally, we have found the HFIP method for Abeta without DMSO less reproducible for our purposes (biophysical studies primarily using NMR) than the ammonium hydroxide method by Teplow. Again, it is likely to depend on the particular question and assay used.

5. HFIP is being used it essential that is removed completely, especially with cells or artificial membranes. See attached paper

Hi Jefferey,

It is exciting to learn about amyloid in perspective of biophysics or other related field. I work on Alzheimer disease using proteomics method like 2D-DIGE. I have read a literature which focused on the toxicity of synthetic and purified Abeta. I hope it will be of help.

Added the prion paper and to the new "In Vivo Imaging" project bench. To access the papers and the discussion, accept the project invitation in the email and check the projects tab on top. Please make sure your email is listed on your profile or I won't be able to add you.

I put together a spreadsheet of all the commercially available fluorescent abeta peptides that I could find. I included the ex/em and links to the vendors.

Please add to it if it is not compleat.

Forgot to add the file

Added bacterial expression papers for Abeta

Dear fellows

I am also working on amyloid beta in C. elegans. I am not sure about my blotting results. I used primary antibody (Ab 3-42) produced in mouse and i got band at 43 kDa. I am worrying whether its working or not? what type of band this is? In papers usually researcher showed bands at 4-25 kDa? is my band correct or something to alter? your kind help is needed. Secondly some body told me to use Tricine-SDS but I unfortunately don't have its recipe.

Abeta oligomers of this size are not that uncommon in SDS gels (see the first attached article). Note that SDS gels may give smaller oligomers than are really present (second article in next post)

SDS gels gives smaller oligomers

Added new papers detailing preparation and characterization of specific abeta oligomers in the cellular biochemistry bench (more to come), differences between brain slices and single cell cultures in the cellular biochemistry bench , details of the NU and 2H4 antibody in the antibody bench (thanks Ian Murray!) and the Shagger von Jagow gel (thanks Ian Murray again!)

Next update: oligomer preparations, nmr techniques, and intrinsic fluorescence for tracking aggregation

Thanks Jeffery! I am interested in fluorescence for tracking aggregation. We are plannning to connect the quantum dot with the amyloid-beta to track the neurotoxic effects of Abeta. I am looking forward to the updates.

Following amyloid aggregation in the presence of inhibitors is a difficult problem. The inhibitors can interfere with ThT binding, which is the most common way of tracking aggregation. It has been found out that the amyloid fibers from a variety of sources are actually autofluorescent and this autofluorescence can be used to track aggregation without additional dyes. The authors use lacmoid binding to alpha synuclein to give an example of how ThT binding gives a false positive for inhibition. It still has to be determined what the molecular origin of the fluorescence is and if other oligomeric forms are also autofluorescent. More details in the "Methodology-Amyloid Fluorescent probes" project bench.

Prediction of amyloid forming segments in protein web servers:

AMYLPRED

http://biophysics.biol.uoa.gr/AMYLPRED/

PASTA

http://protein.cribi.unipd.it/pasta/help.html

FOLDAMYLOID

http://bioinfo.protres.ru/fold-amyloid/oga.cgi

WALTZ

http://waltz.switchlab.org./

TANGO

http://tango.crg.es/

AGGRESCAN

http://bioinf.uab.es/aggrescan/

BETASCAN

http://groups.csail.mit.edu/cb/betascan/

PAFIG

http://www.mobioinfor.cn/pafig/

AmyloidMutants

http://amyloid.csail.mit.edu/

Amyloid kinetic analysis

http://zhanglab.ccmb.med.umich.edu/NEF-model/

Database of current fiber models:

http://people.mbi.ucla.edu/sawaya/jmol/fibrilmodels/

Database of amyloid crystal structures:

http://people.mbi.ucla.edu/sawaya/jmol/xtalpept/index.html

I'll put the original papers up in a bioinformatics section soon.

Semi-automated extraction of lengths, widths, and persistence lengths of fibers from TEM images.

http://www.sciencedirect.com/science/article/pii/S0003269711006646

Video article on preparation of oligomeric ABeta1-42 by the HFIP/DMSO method and measurement of LTP in hippocampal brain slices

http://www.jove.com/video/1884/preparation-oligomeric-amyloid1-42-induction-synaptic-plasticity

Review on characterizing ABeta by several methods including SDS-PAGE, Western Blot and ELISA, DLS, FCS,SEC. ESI-MS/MALDI and capillary electrophoresis

http://www.mdpi.com/1422-0067/13/3/3038

Detection of amyloid forming proteins through extracellular expression in bacteria

(in the Amyloid Prediction Bench)

The authors show how to detect potentially amyloidogenic sequences by the addition of a signalling sequence that exports the protein to the cell surface.Colonies that stain with Congo Red are then selected. The main application would seem to be in finding potential amyloidogenic proteins in genomic libraries. The original research report is

http://genesdev.cshlp.org/content/26/23/2659.full

the protocol is in the link below.

http://www.nature.com/nprot/journal/v8/n7/full/nprot.2013.081.html

High resolution NMR structure of ABeta1-40 in the absence of any detergents or organic solvents (PDB 2LFM) obtained at low concentration (78 uM) and moderate salt . It is not the only structure Abeta can adopt (the protein likely exists as an ensemble of states which can be shifted by a variety of things) but is, as far as I know, the only experimental high resolution structure of low MW Abeta in buffer.

https://www.researchgate.net/publication/51464732_A_partially_folded_structure_of_amyloid-beta(1-40)_in_an_aqueous_environment

Article A Partially Folded Structure of Amyloid-Beta (1-40) in an Aq...

Hello Jeffrey, thanks for this useful information!. I'm looking for methods to stain intracellular amyloid to visualize them by fluorescence microscopy. Do you have any experience with this? Thanks.

FInding small molecules known to bind amyloid beta:

Binding DB database (343 known):

http://www.bindingdb.org/jsp/dbsearch/PrimarySearch_ki.jsp?target=beta-Amyloid+Precursor+Protein+%28APP%29&tag=tg&kiunit=nM&icunit=nM&submit=repeat&energyterm=kJ%2Fmole&column=KI&startPg=0&Increment=50

CHEMBL database:

https://www.ebi.ac.uk/chembldb/target/inspect/CHEMBL2487

While the database is not ideal for amyloid research (for example Ki would probably have to be defined differently) there are several options that are useful including an option to search for similar chemical structures to known binders, known availability through the ZINC database of commercially available chemicals), option to search for bound conformations of the compound to proteins, and calculation of several physical properties.

@Andres

You can try either immunostaining or the chemical probe BSB, a cell permeable Congo Red-like molecule.

BSB: http://www.pnas.org/content/97/13/7609.full

Note that there are additional complications in detecting intracellular Abeta over intracellular Abeta Precursor Protein that are not present with extracellular Abeta.

http://iospress.metapress.com/content/2020755rvmm21444/?genre=article&issn=1387-2877&volume=20&issue=4&spage=1015

See also:

http://cjns.metapress.com/content/c1u4214206580x21/

http://www.sciencedirect.com/science/article/pii/S0024320512000641

I have not tried this myself and would be interested in the experiences of those who have.

Thanks for your help!, regarding to BSB probe I don't know if it works only with Abeta or with several amyloids. Actually I'm focusing my research in bacterial amyloids, so I'll read more about this probe, maybe it can pass through plasmatic membrane of bacterial cells.

Regards

You can also try BTA-1, a derivative of ThT that crosses the blood brain barrier

Sure!, actually we are already using BTA-1 and it cross both the external and internal membrane of gramm-negative bacteria. In fact, it is the best probe we currently have to stain intracellular amyloids. The not so good things are that BTA-1 staining requires previous time-compsuming fixation with PFA, and incubation of bacterial cells with BTA-1 must be done in 100% ethanol, causing cells to form aggregates that can't be fully dissociated after staining (and looks bad in the microscope). Also, BTA-1 stained intracellular amyloids must be observed in the fluorescence microscope using a DAPI-like excitation/emission filter configuration, that in my opinion gives the lowest intensity signal with rapid fading.

So, we are interested in finding new probes for intracellular amyloids!

Almost all of the amyloid fluorophores I know of are in this file

Wow, thanks a lot!

Stefan lichtenthaler has specific Abs to most if not all of the beta amyloid forms. You can use these for staining, I would think. You can contact him via Researchgate. He may know of other tools.

Hi, I am just about to start working with Abeta peptides. Could I store the peptide in chloform, at -20C? For how long?

I wouldn't recommend using chloroform with Abeta, it will form aggregates (not necessarily fibers) with structure different than the native aggregates. See Roland Winter's papers on aggregation of insulin in ethanol and water for example.

Abeta is best stored as dry, lyophillized powder. It can be kept in DMSO for a limited amount of time as an unaggregated sample (how long is up for debate- there is another question somewhere on this site addressing this).

If you are trying to use chloroform to mix it with membranes, keep in mind that Abeta behaves differently if is premixed with the membrane (i.e. with chloroform) or added to the buffer to a liposome solution (see paper below)

If you are trying to get it into membranes without premixing at high concentrations, one method is to first have at high concentration at high pH in 1mM NaOH (no buffer) to a liposome sample. then add concentrated buffer to bring to neutral pH then quickly add the liposomes.

Starting an amyloid project again (after a hiatus working on other stuff):

https://www.researchgate.net/post/Request_for_assistance_and_possible_collaboration_Database_of_the_effect_of_small_molecule_inhibitors_on_amyloid_aggregation

This project page will be receiving more frequent updates in the future (not focused on the database)

First report of the preparation of stable IAPP oligomers using an SDS solution. The oligomers are likely 90 kDa in size, toxic, and unusual in that they are primarily alpha-helical rather beta-sheet.

http://www.nature.com/srep/2014/140304/srep04267/full/srep04267.html

Measuring the amount of amyloid fiber formation quantitatively in tissues. Amyloid fibers are isolated from brain homogenate and then used to seed in vitro aggregation of monomeric ABeta.The change in the kinetics upon seeding reflects the amount of amyloid fibers present in the brain homogenate.

Likely takes some optimization to get it to work.

http://pubs.acs.org/doi/abs/10.1021/bi1013744

I s it possible I can participate in this site and how. Tnx a lot

Hi all,

I don't know whether or not this thread is still active.

But I would have a question regarding pulling-down Aß. basically I am facing to the golden question of IP. How to get rid of the signal of the IgGs ?! Indeed I got a strong signal due to the IgGs that hide the signal of the Aß oligomers.

I know there are some solution like crosslinking antibodies to beads or use of proteins A/G. I would like to know what is the best method.

I thank you in advance for your help/advice !

Benjamin, can you define "signal of the IgGs"?  Crosslinking will help retain antibody to beads, but may result in loss in activity.  The use of tosylated beads is a good solution, but they are a little expensive.  

Signal of the IgGs =  background on a blot due to the presence of the Light and Heavy Chain at 25kDa and 55kDa respectively. I am especially looking to Aß oligomers, thus IgGs background hide the signal of the oligomers.

Benjamin, two bits of advice:

1. Don't use Western blots for assessments of Aß oligomers.  From my own experience and that of others, blots are fraught with reproducibility issues and the induction of artifactual oligomerization (for example see PMID: 23354835).

2. If you forgo the above advice, then you should use protein G-HRP conjugate to detect your primary antibody.  The protein G conjugate will not see the denatured H/L chain in the blot and allow you to avoid non-specific signal.

I would recommend using an assay that is designed for detection of dimer or larger aggregates and that utilizes a buffer system that is as close to physiologically normal as possible (such as aCSF).

A couple of interesting papers on proteomic and genomic studies of Alzheimer's and the connection to amyloid beta:

The first is AlzBase a database of differential gene expression, predicted protein-protein interaction networks, and correlations of gene dysregulation with Alzheimer's severity.

http://link.springer.com/article/10.1007/s12035-014-9011-3/fulltext.html

Database link

http://alz.big.ac.cn/alzBase/

The second is a technique to identify proteins in CSF that bind Abeta protofibers using cysteine coupling to magnetic beads. A few interesting clusters were identified in the immune complement system,hemostasis, and lipid transport

http://pubs.acs.org/doi/pdf/10.1021/cb5008663

This builds on earlier work showing the process of amyloid formation from generic (not native to the organism) amyloidogenic proteins sequester a variety of other proteins. This suggests the protein-protein interactions are intrinsic to amyloid formation itself.

http://www.cell.com/cell/abstract/S0092-8674(10)01372-3

Automated analysis and simulation of transmission electron microscopy of amyloid fibers. Analysis part should work with AFM data (haven't checked, would like to know). Simulation of AFM data would require some modification.

http://www.sciencedirect.com/science/article/pii/S0003269711006646

Interesting question, but I don't think I have the expertise to give a good answer on what the best consensus currently is. Can anyone else help Reyhaneh out?

Is anyone familiar with a suitable cell lines for studying Alzheimers disease?

https://www.researchgate.net/post/Is_anyone_familiar_with_a_suitable_cell_lines_for_studying_Alzheimers_disease#54a0e749d4c118783d8b46bb

Hi ,do you think for studying and research on Alzheimer, rat is suitable model ?the results of rat study can  be extensible to humans due to the complexity of the human brain ???

If you are testing a hypothesis related to a AD  pathomechanism and provided cellular and molecular mediators of your mechanism are similar in rats and humans then, it makes sense to use Rats as model. Anyway, in a strict sense currently we do not have any animal model for Alzheimer's disease. All the current rodent models are models for a mechanism not  disease itself. 

Imran: I'm not sure which quote you are referring to specifically.

Some points though:

https://www.ncbi.nlm.nih.gov/pubmed/21575606

http://www.jbc.org/content/278/13/11612.full.pdf

Hi Jeffrey,

You had earlier written that Genscript peptides are click peptides and are native at pH 7.4. What I want to ask is can we use such peptides directly in aggregation buffers or should we do the HFIP pretreatment to disaggregate pre-existing aggreagtes?

Hi Jeffrey,

I have a question related to a preparation of amyloid beta 1-42 for biophysical study. I use the standard HFIP/DMSO method by Stine et al. and it is ok (more or less). The problem I have is with reproducibility and I've read that it is better to go for NH4OH/NaOH method by Teplow et al. (since it is more reproducible). But the problem is that we don't have a lyophilizer in our lab, so is there any other way to remove NH4OH after introducing it to the peptide? Also, would it be better if I dissolve the peptide in HFIP, remove it with Ar flow and then resuspend it in NaOH and water (instead of resuspension in DMSO/water)? Thanks for the response in advance.

@Asra Nasir Khan

The O-acyl click peptide is not the normal peptide sequence and may have different physical and chemical properties. [1] You can order a click version of the Abeta peptide from Genscript, but this is not the normal Abeta peptide Genscript sells.

Genscript amyloid beta should be treated with the ammonium hydroxide treatment like peptides from other souces.

Article “Click Peptide“: pH‐Triggered in Situ Production and Aggrega...

@Dusan Mrdenovic

2mM NaOH is sufficent to monomerize Abeta [1]

If you want to go the NaOH route, I would dissolve it in 2 mM NaOH in water then add concentrated buffer at higher pH to bring it to the desired pH. A sample calculation can be seen here [2] Keep the Abeta concentration as low as possible even when in NaOH (below ~80 uM is ideal). You can practice with out peptide (the peptide will not effect the pH much).

1Article Ammonium hydroxide treatment of Aβ produces an aggregate fre...

2 http://www.chembuddy.com/?left=buffers&right=composition-calculation

@ Abhishek Jha

• Can I freeze the aggregated solution for further use?

Probably. If the peptide should not evolve further if it is completed aggregated (has completely formed amyloid fibers ). If it is incompletely aggregated (ie a mixture of fibers, monomers, and oligomers) rethrawing will cause further aggregation. The exact amount is hard to predict and not really reproducible. In short, fibers can be frozen other oligomeric species cannot.

• Which method would be more preferable whether keeping A beta at 37 o C for 7 days , HFIP and Ammonium hydroxide based procedure ?

The general observation is that the ammonium hydroxide or sodium hydroxide procedure is more reproducible than the HFIP procedure. See comment to Dusan Mrdenovic above. DMSO is necessary with the HFIP procedure which can also cause many problems in vivo. See

https://www.quora.com/What-effects-does-DMSO-have-on-cell-assays and Adam P. Gunn 's comment in https://www.researchgate.net/post/Problems_with_Amyloid_Beta_Oligomer_Preparation

I perfer to put complex samples into 50% glycerol and store at -20C. Then remove the glycerol on a desalting column or by dialysis or by amincon waiting.

@ Jeffrey R Brender

Thanks for your help. I wonder about few things. I have some peptide films aliquoted and stored at -20C, after HFIP evaporation. I've noticed that if I just take 1 stored peptide film and resuspend in DMSO/water or DMSO/PBS, I already have aggreated peptide. I guess I didn't remove HFIP properly when aliquoting. Therefore, I always add HFIP again and evaporate it with Ar, before I proceed (to try to monomerize it). I'm not sure for how long should I keep amyloids in HFIP before I evaporate it and proceed with preparation? So far, I've tried leaving the film in HFIP for 30, 60 min. and 24h and I couldn't get only or mostly (even though I use ultrafiltration with 10 kDa filters at the end) reproducible results.

If I go for NaOH, should I add HFIP and evaporate it before I add NaOH or just go with NaOH/PBS? I ask this because I know that If I pass through peptide's pI, aggregation rate will increase.

Thanks once again.

By the way, I want to study amyloid beta 1-42 interaction with lipid bilayer using AFM. I want to see what will monomers do to the bilayer (oligomers as well). So, this is the reason why I would like to get as homogeneous sample as possible. Also, I would appreciate if someone share some papers related to the amyloid 1-42 monomer and oligomers size.

I hope our finding about HFIP treatment may help many Abeta researchers. Three conclusions.

(1) Anyway, DON'T BELIEVE THE HFIP TREATMENT TOO MUCH.

(2) Conbination of evaporation (SpeedVac) and HFIP treatment must be avoided.

(3) Lyophilization of dilute HFIP solution of Abeta is better. Remind the concentration of Abeta during HFIP treatment.

Article Nuclear magnetic resonance evidence for the dimer formation ...

Does anyone have information about the lyophilization of amyloid beta? What kind of conditions should be achieved to properly lyophilize amyloid beta 1-42 powder in NH4OH? Thanks.

Dusan Mrdenovic " I want to study amyloid beta 1-42 interaction with lipid bilayer using AFM. I want to see what will monomers do to the bilayer (oligomers as well). So, this is the reason why I would like to get as homogeneous sample as possible "

Definitely do not use HFIP in this instance as it destabilizes bilayers.

See Article Amyloid-??-Induced Ion Flux in Artificial Lipid Bilayers and...

" I would appreciate if someone share some papers related to the amyloid 1-42 monomer and oligomers size "

The classic paper on Abeta ion channels is Article Amyloid ion channels: A common structural link for protein-m...

most papers looking at ABeta effects on membrane will cite this paper. See also the Justin Legleiter group and some of the papers in my former lab in the https://www.researchgate.net/project/Biophysics-of-amyloid-aggregation-and-potential-amyloid-inhibitors project

On the subject of the Abeta monomer, see

Article A Partially Folded Structure of Amyloid-Beta (1-40) in an Aq...

hi friends

i am testing the compounds on beta amyliod and facing alot of problems ,

1) i dont know why in my assay flourances values decrease....

2) i am too much confuse about the buffer and its pH mean which buffer is best and pH?

can any one share protocole ....because i am not satified with my present protocole

@ Jeffrey R Brender can you tell me how we calculate the % inhibition of compounds beta amyliod assays ,,,,,

Regarding the lyophilization protocol of amyloids - Here is the answer that I've got from Timmothy Ryan, the author of the paper about ammonium hydroxide preparation method of amyloid beta (DOI 10.7717/peerj.73):

"Hi Dusan,

We just used the highest vacuum setting that the lyophiliser could provide, and no heating; generally I would freeze 100-200 microlitres of the NH4OH/Abeta42 solution in eppendorf tubes using liquid nitrogen (leaving them for about 30 minutes to make sure that they were solidly frozen), then used the freeze dryer with the relatively default settings. Typically it took overnight to fully dry the peptide out."

https://peerj.com/questions/3495-amyloid-beta-lyophilization/#annotation-3498

Hi guys,

I wonder what is a reliable way to check for residues of NH4OH in amyloid samples? I freeze dried amyloid 1-42 with 1%NH4OH and now I would like to check if all of NH4OH is gone. Thanks.

BR,

Dusan