Hi Apurva,

I just wanted to point out that HFIP 'monomerisation' of Abeta is widely used due to tradition in the AD research field, not because it's necessarily the best method. It essentially stems from the use of acids to dissolve plaque material in the early research days. HFIP is a beta-sheet disrupting solvent, so it is supposed to break apart fibrillar Abeta, but it can also induce oligomer formation in its own right (http://www.ncbi.nlm.nih.gov/pubmed/?term=pachahara+AND%20HFIP).

We routinely use NaOH to dissolve Abeta by bringing it to high pH, then neutralising with PBS. David Teplow wrote a great paper on this method a few years back (Teplow, 2006, Methods in Enzymology 413, pp 20 - 33). He (and I) argue that dissolving Abeta in acids then bringing to neutral in PBS (or cell culture media) will cause the peptide to pass through the pI and form oligomers/aggregates. You won't be able to see these on SDS-PAGE as they will break down in gel conditions, but solution based analytical techniques (e.g. analytical ultracentrifugation) will show that there is an abundance of oligomers. Dissolving the Abeta in NaOH/PBS also means the peptide can be quantified easily by UV spectrometry (we use an extinction coefficient of 94,500 at 214 nm, but this will vary a bit from batch to batch).

Another reason we avoid HFIP/DMSO is because DMSO quenches radicals and thus silences the redox activity of Abeta (in the presence of metals such as Cu). Using metals you can rapidly generate a range of oligomers - i.e. in the space of 30 min, which is much faster than making ADDLs, amylospheroids, globulomers or any of the other reported 'soluble oligomers'. I've attached a blot of these Cu-induced oligomers that were separated by electroelution.

Have you ever asked the question what biological process you try to model by this protocol?? Lot of people in lot of laboratories have succeded to get oligomers and probably lot of people have seen some bands in SDS (we also have), but what does it mean? does it confirm that the solubility limit of abeta is 10 micromolar or that SDS can induce the trimers as an artefact??

You dealt with a very tricky staff and I should blame on myself too, that’s why the amyloid story in AD is still under debate.

Anyway, I will be happy if it will help you:

1) One should completely dissolve amyloid powder, need 50-60 min, sometimes need to be at 37C for 15-20 min, even if solution looks clear.

2) Split in necessary volume aliquots, you should treat all of them at once as described on next step.

3) Under the ventilation hood!! You should completely evaporate all traces of HFIP, the qucker – the best, use the nitrogen flow (very slow, use 200uL yellow tip placed on the end of the silicon tube from nitrogen source!) directed toward the top of the opened eppendorf tube with amyloid solution. In my lab it looks like giant millipede.

4) After you add DMSO, one need to sonicate solution on the water bath for 3-4 min.

5) Prepare necessary volume of 0,1x DPBS (dilute ordinary one by ten times) in siliconized eppendorf tube. Acidic conditions will increase the Oligomer size, whereas the most usefull (from tox point of view) ones should be 11-20 mer oligomers.

6) The best results if the final Ab42 concentration (in polymerization solution) will be less than 90 uM.

7) Add Amyloid – DMSO solution drop-by drop(3 uL each) in to 0,1xDPBS polymerization solution, while slowly shaking the bottom of the tube (do not Vortex!)) by finger. Finally, mix the closed tube by inverting end-over-end.

8) Sonicate the tube for 5 min at RT.

9) Place at 37C for 2 h and then at +4C for 24h.

10) Next day- centrifuge at not more than 5000g for 20 min at +4C.

11) Carefully remove the supernatant by leaving around 25-30 uL in the bottom (even if the pellet is invisible) . Keep pellet fraction for SDS-Tricine-PAGE electrophoresis.

12) Take 5 uL sample from top and 1 uL of bottom fractions and run 10-20% gradient SDS-Tricine-PAGE electrophoresis.

13) Fix the gel with Meth-Glacial Acetic Acid solution and stain with either Silver or even better use the Copper-based Stain kit from Pierce.

14) I f all above doesn’t work, then let me know, we will try micelle-based approach next time.

Good Luck!!

Sergey

Let me try these few steps.. which seems to be promising... will come back... !!! Thanks...

Two things you may want to consider:

1. Leaving the tube overnight is not enough to remove the HFIP. Trace elements will remain and cause problems both with excess aggregation and artifactual toxicity (see Capone et al Neurotoxicity Res 16 pg 1-13)

The HFIP must be removed by a nitrogen stream or lyophillized to remove it completely. 19F NMR with an internal fluorine standard like trifluoroethanol is a good way to check for residual HFIP.

2. If further removal of HFIP does not work, try using a 1% ammonium hydroxide solution or 1 mM NaOH as an additional step or instead of the HFIP step. This breaks down the AB more fully. However, it may not give you the type of oligomers you want.

3. IF none of the above work, try lowering the concentration of the stock solution. AB has both a nonlinear concentration dependence and hystersis. There are at least 3 critical concentrations around 1, 17 and 200 uM in buffer that lead to different behavior of the peptide and are not fully reversible. (Fawzi 142, 9948 and Sabate J. Phys. Chem B 109 11027). I don't know if similar behavior exists in DMSO.

You may want to try the ADDL prep method as described by Klein (2002). They use F12 as their redissolving media rather thanPBS, but other than that it seems like a very similar procedure. I agree with a previous poster that you should use more than simple overnight evaporation to get rid of HFIP. If you have a vacufuge or a speedvac, that would work, or you can use nitrogen flow.

Also, the smear you are seeing in SDS-PAGE may be the result of a diverse distribution of oligomer sizes. AB is only 7.5 kDa, and we often see oligomeric species appear as a smear with a molecular weight range, rather than a distinct molecular weight.

If proteins are being precipitated out of your solution upon centrifugation, they are probably fibrils, rather than oligomers. Have you run SDS-PAGE on the pellet to see what molecular weight species are being spun out? If you're getting a great deal of fibril formation at 4 C over 24 hours, you may want to decrease your incubation time. Also, we have seen that low concentrations of DMSO can significantly accelerate aggregation, and it is well known that acidic conditions cause aggregation. We do a great deal of our work at pH 8.0 in TRIS buffer, which often helps to make lag times more workable.

As for protein degradation, I'm not sure. We never store our aqueous AB preps in the -80, because we worry that they will be harmed in the freeze/thaw process. Generally, we keep our monomer/oligomer preps in the 4 degree, where they can sit for three days to a week before aggregation really gets going.

Hope this helps.

Another two things to consider

1. PBS instead of F12 culture media primarily gives the larger oligomers called amylospheroids (40nm across) and short fibers instead of ADDLs (4 nm)

(J. Biol. Chem. 278 11612–11622, 2003, Nat. Struct. Biol 14 1157)

2. SDS-PAGE can give artificially small oligomers by splitting large oligomers . It would be good to try a native gel as a comparison (Biochemistry 2006, 45, 15157-15167or another complementary technique such as NMR DOSY or light scattering

@Lisa Stone: The smear had end just above 6.5 kDa marker band...

I agree that the DMSO step should be removed. We generally lyophilize the protein after removing HFIP. The lyophilized protein is then dissolved in a buffer.

You are following a standard protocol, but keep in mind that a 24 hour incubation will result in significant fibril formation. There is a steady state between the monomer, soluble oligomer (multiple species) and fibril aggregation states. The soluble oligomer population is a small percent of total amyloid beta in your preparation. The smear on your gel is likely due to a many different sized oligomers(12-mers, 24-mers and such). You can try to do a time course experiment (T 0, 2h, 4h, 8h, 12h and 24h) ti find optimal incubation period. Unfortunately, prepared soluble oligomers are not stable enough for storage.

I suggest you review the early literature for tubulin assembly to microtubules (1970's). While the circumstances of assembly for Abeta are distinct, those studies show assembly intermediates are often the retsult of non-physiological nucleation.

My procedure was simpler; i observed bands at 4k and 8k.

1. Monomer formation

- dissolve powder in DMSO at 1 mg.mL-1

2. Dimer formation

- further diluted in PBS at 0.1 mg.mL-1

- incubated for a week at 4 °C

Thanks everyone for your suggestions... Fortunately, I have been able to get a much better yield this time. I ran a tricine- SDS gel which is shown below.

The LAST FOUR LANES (the highly black ones) contain the samples:

A. Protein at 0 Hour, just after making 100 uM stock;

B. Protein just before centrifugation after 24 hours at 4 deg C;

C. Protein after centrifugation after 24 hours at 4 deg C;

D. The pellet after centrifugation was dissolved with 50 ul of 1X PBS.

The gel run was 10% tricine SDS- PAGE. The protein load was 1ug.

Please suggest me on how can I improve on this now. Do I need to run higher percentage gels to resolve them better.

Be aware that SDS-page can give artificially small oligomers, SDS splits larger oligomers into small ones. A native gel without SDS would be better to characterize them

I have a related question about Abeta preparation : I use a reverse (42-1) peptide as a negative control, which I prepare in parallel with my 1-42 peptide, and then I test their toxicity on my favorite cells at various concentrations. BUT the reverse peptide seems to be equally toxic! Has anyone had similar problems? Could it be leftover HFIP that is toxic? I let it evaporate under N2 flow, but maybe there are HFIP traces left, or impurities?

Residual HFIP is toxic. HFIP can be tested either by 19F NMR or derivitization and HPLC. Evaporation by N2 flow is not sufficent but lyophillization or N2 flow + vacuum overnight will remove it.

If you are able to rule out HFIP-induced toxicity, it would be quite interesting if your reverse peptide is indeed toxic! Have you tested it to see if in any way it is capable of aggregation?

Thanks for your prompt answers.

Jeffrey : I'll definitely use a vacuum overnight the next time I prepare my peptides.

Matthew: I need to repeat the experiments. Unfortunatelymy lab is not equipped to check that : we only look at Abeta aggregation by Western blot and we don't have antibodies against the reverse peptide.

Just want to know, can we use IPA instead of HFIP in the 1st step of dissolving the amyloid peptide? What is the reason for using particularly the fluorinated isopropanol.

Hi Apurva,

Let's make right things step-by-step from your original protocol, hope we are talking about Ab42, not Ab40(!?):

1. 1mg of amyloid pure peptide is dissolved in HFIP (400 ul) and is incubated at RT until clear solution forms.

it might take 1-2 h! One can warm up at 37C to speed up the process.Important: Source of the peptide - Bachem is best and try TFA salt instead of HCL.

2. Aliquoting the solution in to 4 tubes with 100 ul each.

I would split into 50 uL aliquotes , important to use siliconized tubes!

3. Storing them at -80 dec C except one of them which is left open in fume hood overnight to evaporate HFIP and make the peptide pellet dry.

Wrong! All aliquoted tubes should be placed under N2 flow into each one! Be carefull: the gas flow should be very weak otherwise you can spill out the solution easily! Only after complete evaporation of HFIP, the tubes should be tightly closed and placed at -70C. The dried peptide should be invisible on the tube wall. If there will be milky dots or spots - repeate the solubilisation in HFIP(using the same volume) and evaporation!

4. Next day, 5mM stock of Abeta is made using DMSO (~11ul; according to calculations).

Important: use only freshly opened DMSO, prefrearbly tissue culture tested (Sigma)best to use it from 10 ml ampules.

5. The peptide at the bottom is dissolved by DMSO and then transferred to new tube to which around 540 ul of 1X PBS (freshly prepared; pH- ~7.4-7.48) is added to make 100 uM ABeta PBS stock.

Important:

i) Tube should be siliconized!

ii) PBS should be prediluted to 1:10, as the NaCl and high phosphates also not good, I filtrated it before through 0,22um syringe filter to avoid any crystall or anything else present.

iii) DMSO should be added drop-by drop using ultrafine siliconized tip. Do not use Vortex to mix, just tipp by finger.

iv) Final concentration of amyloid should be 100kD bands of protofibrills.

Appreciate if you let me know your progress.....

Hi Apurva,

I just wanted to point out that HFIP 'monomerisation' of Abeta is widely used due to tradition in the AD research field, not because it's necessarily the best method. It essentially stems from the use of acids to dissolve plaque material in the early research days. HFIP is a beta-sheet disrupting solvent, so it is supposed to break apart fibrillar Abeta, but it can also induce oligomer formation in its own right (http://www.ncbi.nlm.nih.gov/pubmed/?term=pachahara+AND%20HFIP).

We routinely use NaOH to dissolve Abeta by bringing it to high pH, then neutralising with PBS. David Teplow wrote a great paper on this method a few years back (Teplow, 2006, Methods in Enzymology 413, pp 20 - 33). He (and I) argue that dissolving Abeta in acids then bringing to neutral in PBS (or cell culture media) will cause the peptide to pass through the pI and form oligomers/aggregates. You won't be able to see these on SDS-PAGE as they will break down in gel conditions, but solution based analytical techniques (e.g. analytical ultracentrifugation) will show that there is an abundance of oligomers. Dissolving the Abeta in NaOH/PBS also means the peptide can be quantified easily by UV spectrometry (we use an extinction coefficient of 94,500 at 214 nm, but this will vary a bit from batch to batch).

Another reason we avoid HFIP/DMSO is because DMSO quenches radicals and thus silences the redox activity of Abeta (in the presence of metals such as Cu). Using metals you can rapidly generate a range of oligomers - i.e. in the space of 30 min, which is much faster than making ADDLs, amylospheroids, globulomers or any of the other reported 'soluble oligomers'. I've attached a blot of these Cu-induced oligomers that were separated by electroelution.

I would agree with Adam, albeit with certain remarks:

NaOH -solubilized amyloid preparations gave me also oligomers with simillar MW, although they demonstrated different biological effect on both neuronal and non-neuronal cells. The Cu-induced oligomerization is well-known and will also gave another type of oligomers (in terms of both bilogical effects and biochemical properties), albeit of simillar MW.... The MW simillarity is not a valuable parameter to compare different Oligomer preps.The key Q - what do you want to achieve with such Oligomers...???

its better to keep the dry HFIP films from step 1. Kept dry, they seem to last for years.

Freezing may cause additional aggregation. When the sample is frozen, the effective concentration (thermodynamic activity) of water decreases because the ice crystals are no longer interacting with Abeta. Consequently, the effective Abeta concentration increases. This happens to lesser extent even when it is flash frozen and the same process happens when it melts.

For Theanmalar Masilamani question:

Fluorinated alcohols are essential. Non-fluorinated ones do not work. The fluourinated alcohols break up apart the fibers by stabilizing an alpha-helical monomeric conformation of the peptide, while non-fluorinated alcohols seem to stabilize amorphous aggregates. The exact reason why fluorinated alcohols stabilize helical conformations is disputed but some theories are given as references 58-63 in the paper below.

The strength of breaking up aggregates is roughly TFA>HFIP>TFE, but TFA is not preferable because of its tendency to cause other problems with the peptide.

Regarding NH4OH vs HFIP:

DLS, ThT, and SAXS show HFIP treatment of Abeta42 apparently leads to faster amyloid formation, a heterogeneous mixture of large oligomers, and less toxicity when compared to ammonium hydroxide pretreatment.

The review of the article can be found here:

https://peerj.com/reviews/73/

https://peerj.com/articles/73/

I do not think it is wise to use PBS, use F12 culture medium instead. Concentrations of salt influenced the aggregation of Abeta1-42 significantly. You may refer to this paper"In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis"(J Biol Chem). I once made the same mistake like you. In PBS, I did not get enough Abeta1-42 oligomers, however, in F12 culture medium, the deposition at the bottom of tubes after centrifugation was much smaller. Good luck!

F12 media has been reported to give a higher yield of oligomers than PBS. This is probably indeed the reason for the low yield in Apurva's original experiment. It is difficult to do certain biophysical assays in F12 though (CD and NMR, for example)

I see. However I did not get enough oligomers in PBS as in F12 media. Is there a solution for this ? I suggest Apurva try ddH2O. It might work

F12 is not a problem for most assays, just some of them. The oligomerization appears to be concentration dependent. PBS will apparently give some oligomers if the concentration is between than 20-50 uM (Fig. 3 in the paper below). Lower than 20 uM neither oligomers or fibers will form at 4C without shaking. At 50 uM and above only fibers are formed after 24 hours. This finding suggests the 100 uM concentration is too high.

We think we know the physical basis of this effect; I am in the process of writing the paper now.

I personally would be very, very interested in protocol that gives oligomers at high concentration in PBS or other non-cell cuture media, or if anyone knows if oligomers formed in F12 can be reconstituted in PBS.

http://www.jbc.org/content/287/29/24765.long

That would be an interesting research.

Hello everyone,

This thread has been really helpful in designing my own experiments. I am also working with amyloid peptides and have been trying to characterize oligomers and aggregates on a gel. The dilemma that i have been facing by reading the vast literature is that whether SDS-PAGE is an appropriate method for detection of oligomers? Whether NATIVE-PAGE is a more suitable method for detecting oligomers and HMW aggregates? I have run a couple of NATIVE gels for my samples and have got bands in the range of around 20kDa but to differentiate between monomers and oligomers i will need a marker with lower kDa range.

Any help in this regard will be very helpful.

Thanks

SDS-PAGE is probably not an appropriate method since SDS splits larger oligomers into smaller oligomers. Native gels have less of a problem; however

1. Protein-protein complexes can dissociate in the gel and will elute as a single peak if the exchange is rapid. A similar problem exists with size exclusion chromatography (Biochemistry, 2012, 51 (17), pp 3694–3703).

2. If your aggregates are very large they may not enter the gel at all

http://pubs.acs.org/doi/abs/10.1021/bi061850f

so what is the protocol for making the abeta oligomer? I always can't dissolve the abeta with HFIP. Could anybody tell me why?

I am not sure the best place to add this answer, however this is an anecdote that I think is important to share. This may also be in the literature as well but I do not recall coming across it. Simply put, during purification of abeta I have found that addition of a low percent of of albumin greatly reduces loss of the peptide. I have been trying to purify material for mass spec and cell toxicity assays and until I added albumin into recovery steps I found (or actually didn't find) the peptide.

What is the concentration of albumin that you have added? Can you please describe this more in details? I would very appreciate it

Ella, I have used anywhere from 0.05 to 0.005% depending on the downstream use. I also have be "preblocking" tips and tubes to avoid loss. One day I was called away from the bench for an hour and the abeta that I had just put into a new tube following an IP elution was lost, whereas the sister IP elution I did when I returned was fine. This started to clue me into the fact that adsorption to even "low-binding" tubes can be quite rapid without a buffer protein.

I started a project here to share protocols

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

you are welcome to add anything you want

As far as albumin goes, Abeta has a fairly strong direct interaction with HSA in the micromolar to submicrolar range

Stoichiometry and affinity of the human serum albumin-Alzheimer's Aβ peptide interactions

http://www.ncbi.nlm.nih.gov/pubmed/21190670

See

Mapping the interactions between the Alzheimer's Aβ-peptide and human serum albumin beyond domain resolution.

http://www.ncbi.nlm.nih.gov/pubmed/24094411

Human Serum Albumin Can Regulate Amyloid-β Peptide Fiber Growth in the Brain Interstitium

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431649/

Understanding the molecular basis for the inhibition of the Alzheimer's Abeta-peptide oligomerization by human serum albumin

http://www.ncbi.nlm.nih.gov/pubmed/17367135 (as well as other papers by Giuseppe Melacini )

HSA also binds and inhibits other other intrinsically disordered peptides like IAPP

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0069652#pone-0069652-g004

@Xiabo: To dissolve abeta peptide, follow these steps:

1. Add 400ul of HFIP to the vial containing 1mg peptide.

2. Vortex it for some seconds.

3. Leave the vial with HFIP at room temperature 2-3 hours with vortexing every hour or so.

4. Generally, at the end of 2 hours the abeta peptide does get dissolved in HFIP.

5. If you still see some speck of peptide floating in the solution, keep it for another hour.

6. If something still remains insoluble, I guess that's some impurity.

Which company do you buy abeta peptide from and what is the purity/ peptide content in 1mg (as mentioned on the certificate of analysis)?

Could you show your steps you finally use?

@Fangqiang: Oligomeric amyloid beta was successfully prepared using the same above mentioned protocol.

Apurva, Thank you for response. I have another question. Does the oligomeric Abeta cause primary cortical neuron cell death in your hands? 

I didnt test them on primary neurons yet. But my senior who standardised this procedure tested them on hippocampal primary neurons and she successfully observed cytotoxicity. I tested on hippocampal cell lines and it does cause cytotoxicity to them as well... 

Again, I will pose the question to those using synthetic material:  what is your goal and how does this relate to disease??  Dosing micromolar quantities of abeta-42 oligomers to look at cortical death?  What does this recapitulate?  

Hi guys

I struggled to get a reproducible results from thioflavinT assay but every time is something different and the standard deviation is huge. I bought AB (1-42) peptide from American Peptide company (USA) and did the following protocol:

 1. dissolve 1 mg AB in 1ml, TFA  then vortex and sonicate for 15 min then add another 1mL.

2. evaporate by dry N2 then remove TFA by using 10mM HFIP 1mL and evaporate 

3. I repeated the process of adding HFIP three times and finally remove any traces of HFIP using freeze dryer for 2 hours.

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

The problem is when I mix my compounds to test their disaggregation ability on AB by using ThT-T assay, the results are not reproducible and this makes me feel sick. 

After converting the peptide to monomers, i keep them in tubes in the freezer (-20C) and used them when required. I do not know what is the problem and why it gives such a different results

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Any idea will be extremely useful.......Thanks

As the other posters pointed out, HFIP is not a great solvent for disaggregating Abeta 42. NaOH or ammonium hydroxide work better. 

Does 10 mm HFIP mean 10 mm HFIP in water? If so, you need to use 100% HFIP. You can skip the TFA step. It has been known to cause posttranslational modifications in some cases. HFIP is surprisingly hard to get rid of. Try freeze drying for 24 hours instead of 2. Finally,  if you use the HFIP procedure, the final step is dissolve the fibers in DMSO.

However, DMSO causes problems with both cell and biophysical assays. The NH4OH/NaOH procedure we found is both more reproducible and more compatible. 

1. Dissolve the peptide in 1%NH4OH at 1mg/ml

2. Freeze dry for 24 hours

3 Reconstitute in 1 mM NaOH at 90% the concentration. Add 10x buffer to the final concentration.

4. Run the solution through a filter. We usually use a SpinX centrifuge filter. This step is usually not necessary but the loss is minimal if a centrifuge filter is used. 

The quality of the monomerization procedure can also be checked. In a UV-VIS the tyrosine peak should be distinct.A spectrum looking like an exponential curve is an indication of Rayleigh scattering from aggregates. In a 1D NMR the amide peaks should be recognizable and the peaks relatively sharp. Missing or broad peaks is an indication of slow binding between the monomeric peptide and oligomers in solution. 

hey

after you make 5mM Abeta stock in 100% DMSO (this should be fresh ie 20ul of DMSO in 0.45g of abeta) add that in F12 medium (ie dilute it) take 20 ul of Abeta in DMSO stock and 980 ul F12 media. Incubate the solution in 5 degrees for 24 hours. then centrifuge at 14,000g for10 mins in cold. then supenatant shall be your oligomers!

Hi,

Does the NaOH method work for lysophilized Abeta 1-40?

Hi,

Is amyloid beta easily filter  from whatsman filter paper .???

@Syed No. The pore sizes (~2-20 um) are too large and will let aggregates get through

@Benson Yes. The NaOH works on Abeta1-40

Hi,

Does the NaOH method work for lysophilized Acetyl-Amyloid β 25-35?