Hi, 

From what you have said I would:

1. Check the detector gain, and modify to maximise the signal (without going over measurement maxima)

2. Agitate more vigourously and/or for longer periods of times between measurements.  I suspect the fluorimeter does not take a reading during your measurements.  Using a BMG Omega plate reader I have found orbital agitation at 500 rpm for 5 minutes maintains preformed fibres in solution (to around 80% over the time period you are measuring).  My colleagues use a 300 rpm figure of 8 orbital for 10 seconds before a measurement to measure the fibrilisation rate.  I suspect your primary problem is settling of the fibres in your samples, hence a very low erratic measurement.

I have two further suggestions :

1. You could also play around with the excitation or emission slits to get more intensity out.

2.  Usually 446nm is used for excitation and you look at 484nm. Are you using these?  

Hi,

Before anything are you sure for your comment "I'm using 200mM ThT concentrations to monitor 20-50uM abeta"

200mM ThT is too much 

What is your ThT concentration? 200 mM ThT doesn't sound right. Peptide preparation protocol and aggregation conditions look fine. Check the fibrils by EM, maybe you're making oligomers instead. 15 seconds agitation sounds too short. Also, you can add a teflon or plastic ball to each well, it will make agitation more efficient.

Yes, 200 mM is too much. probably Tht is already forming micelles. I never used more then 20 uM. I don't think the dye is even soluble at that concentration.

if 200 mM of ThT is not a spelling mistake, then it may be the main problem.

Else there is a number of factors which can influence ThT intensity - using different shaking, different plates, different abeta preparation or different batch of peptide (especially if sythesized by different company) etc.

Another thing:

Use a buffer at a pH of 9 (e.g. glycine) in your cuvette for measuring the aliquots (obviously NOT during your aggregation experiment). Maximum fluorescence for ThT is observed at pH higher than 8 (see Naiki et al, Anal Biochem 1989)

200mM Thioflavin T is way too high ! A stock concentration is usually 10mM and used at 10uM in a reaction. This high a concentration will produce an inner filter effect reducing the effective intensity of exciting light. This alone could account for your low fluoresecence readings.

Also check machine parameters, scan for optimum excitation and emission wavelengths and then widen both wavelength bands around the optimum to produce the required intensity. Check any attenuation or gain settings as well.

I'm sorry for the confusion. I meant to type 200 micro molar (uM). I know that even this is high and after reading everyone’s comments I'm switching back to the 20-30uM ThT. I was following the protocol provided by Anaspec for their AggreSure kit, but after reading the attached article I think, in addition to others, a 1:1 ratio of Tht:ABeta should be all I need. 

@Peter John Davis: Is the type of agitation you are mentioning acceptable and common practice. I was not agitating before and fibrillization took a week or more. I'm worried about agitating for two reasons. One, I'm going to be comparing the assay results with AFM images, and I cannot agitate on the AFM in solution. Secondly, my modification of the peptide is subtle and I don't want to be breaking up fibrils seeding reactions that would otherwise override any inhibitory effect.

Article Thioflavin T Promotes Aβ(1–40) Amyloid Fibrils Formation

I'm going to second checking the gain on whatever detector you are using. Sounds like it's too low. Noisy signals and desperation are common when working with our ol' friend Abeta! :)

Also glad you are dropping your concentration down. This will help immensely.

Carefully hold the microplate to the light at the end of the incubation taking care not to make any sudden movements that might disturb sedimentation . If you see a yellow precipitate at the bottom of the well, the fibers are sedimenting out during incubation. Sedimentation can causes a drop in intensity as the fibers are no longer at the focus of the light beam. Unfortunately, I don't know any solution to this problem besides shaking.

An approximate 1:1 ratio is commonly used.  I tend to maintain a constant 10uM final concentration in all my experiments to allow ready comparison between experiments.

The most 'reliable' means of comparing the absolute polymerisation kinetics to the AFM experiment is to perform a discontinuous assay, whereby you have a container of Abeta diluted in your buffer etc. incubated at x degrees C.  At specific times remove an aliquot, add to the cuvette or plate which contains a defined drop of ThT and then quickly measure in a cuvette/plate reader.  The pipetting acts to mix any fibres/ThT and this method ensures that the mixing/ThT is having a minimal effect on your results.  The downsides include:  high Abeta mass required, limited sampling due to the peptide mass used, noisy data due to the inherent errors in pipetting, timing and general nature of the experiment, evaporation of the abeta preparation changing the concentrations of reactants and is quite time/sleep consuming.  However it is probably worth collecting a decent data set this way once to justify everything in the experimental set-up, although the data will probably be quite noisy in comparison to the plate reader automated data.

As for shaking, gentle agitation is quite often used.  I would recommend minimising agitation as this typically increases the frequency of  nucleation site development/elongation rate/fibre fragmentation.  Try 10-30 secs at 300-500 rpm before reads and have a slower sampling rate of perhaps 5-15 minutes.  These values are all instrument dependent of course.   

Hey Guys. I have the same problem. My protein does not aggregate recently and I have tried everything to put it back to normal, but efforts are useless.

I think 10 to 20 uM of ThT is sufficient.Also, if working with a plate reader, you can increase the slit width.

Hello everyone

We are trying to form fibrils of Abeta42, but we are not getting any increase in fluorescence intensity even after 48h of incubation at 37 oC  at 250 rpm, 

Final working concentration are: Abeta (4 micro molar), ThT (10 micro molar) in PBS buffer (pH= 7.4) to a final volume of 100 micro liter, (fluorescence recorded with a plate reader)

Secondly, the fluorescence intensity keeps fluctuating among the duplicate wells even.....

Anybody please suggest what we can do...Thanks...

4uM is a low concentration of beta amyloid for ThT assays, so I'm not surprised you don't see anything for that long of a time period. That is not to say that 4uM beta amyloid assays are not doable. I would suggest looking at this paper in the link below because even at 20uM without shaking my ThT assays take 72hrs, and with shaking they were previously not reproducible among triplets. I started using this technique of adding borosilicate glass beads to the plate before this paper came out and was able to get very very reproducible results even across multiple batches, and was still able to observe the effects of my variables. So the paper is applicable to beta amyloid.

Prep is also very very important in beta amyloid experiments. I suggest an alkaline prep over HFIP/DMSO (Stein prep), there are tons of papers using either so that is up to your experimental setup and limitations.

http://www.sciencedirect.com/science/article/pii/S0006349516342783 

Hello Deepti..

Are you doing it the first time? or the same experimental set up gave results before?

Also what kind of 96-well plates you are using and where are you reading fluorescence from? i mean bottom or top?

Sometimes these details also effect the readings.

 Thanks Albert for your reply and useful suggestions, Can you please tell me what is the workingconcentration you use for ThT for 20 micromolar Abeta.

Hello Shreyasi. we did on same concentrations earlier but with different spectrofluoremeter, that time we got increase in fluorescence in 21h, this time we shifted to another instrument where we can attach plate reader, and observed no change in emission, intensity remains below 200. We are using black wall well plate.

Thanks a lot...........

Please check the slit width of the reader.  You can increase it if you are observing too less fluorescence.  I ASSUME THAT YOU ARE USING A 96 WELL PLATE. WHERE IS THE INCIDENT LIGHT COMING FROM?  Top or bottom? 

Hi Deepti,

The absence of ThT fluorescence in amyloid assays can be caused by many factors, which might not be mutually exclusive. As Albert mentioned before, 4 uM Abeta concentration is a bit too low, but still, you should be able to detect some signal. Thus, you must be first sure that the lack of signal is due to the absence of Abeta fibrils (meaning that you don't generate any fibrils during the 48 hr period, and that is why you don't get any signal). Transmission electron microscopy (TEM) would tell you the answer. Run the assay, collect your post-assay solutions, and analyze them with TEM. If there are plenty of fibrils, then either your ThT solution is not good, or the aggregation assay buffer is somehow contaminated (e.g., metals), or the Fluoroskan machine has some issues (lamp, filters, shaker, temperature controller etc.). Any of these factors can be the real culprit!

If you did not observe any fibrils with TEM, then double (or triple) your peptide concentration, and see how the signal changes (and check with TEM as well). You can also increase ThT concentration to 20 uM. 

Also, check the purity of your Abeta sample; do you synthesize it or you purchase it? Make sure it is absolutely pure (mass spec is the best way to check that). 

Hope this can help.

Deepti Goyal,

I always make my ThT solutions in a 1-1.5 molar ratio to that of the peptide. There is a paper, that I can't find at the moment, that suggest this results in the formation of ThT micelles. However as Abdolvahabi has suggested it's important to test the solutions with something like TEM or AFM (AFM being what I use) post-assay. As such I have never observed such micelles. and a 20uM concentration of ThT is pretty standard in papers carrying out such assays. 

Thanks everyone for the suggestion...

@ Abdolvahabi we bought Abeta 42 from AnaSpec and Sigma Aldrich, and we make the peptide stock in 10mM NaOH.

I will see all the possibilities and work on them, that you have suggested.....

Albert Pilkington 200 mM ThT is too much! I am sure at this much concentration of ThT, you will not observe a reasonable fluorescence due to the 'inner filter effect'. A starting concentration of ThT in your reaction should be 20-25 uM. If you want to delve deep into this problem, you should plot the fluorescence intensity of ThT with respect to its concentration and you would get a reasonable range of ThT where your intensity vs concentration curve is linear.

200 mM of ThT is too much, plus it's better if you optimize the ratio ThT[protein concentration used.