I have doing ThT fluorescence (one point and time kinetics) on abeta peptides. My peptide is abeta 25-35 fragment. I am dissolving 1mg of it in 50ul 100% DMSO and then adding 225ul h20 and 225 ul DMEM buffer. final volume of this mixture is 500ul. I store this solution in 4 degrees. after 24 hours, I take it out.
I take out 110ul of this samples add 10ul (100uM ThT) and add 120 ul of this mix into a well of 384 well black plate (Nuanc) to take one point ThT fluorescence reading. I have also done experiments with other buffers and conditions. the observation I make with DMEM is that it has high fluorescence at 500-550. I compared this data with other conditions and did not find such observation.
Please kindly help me out what is going with abeta 25-35 fragment in DMEM compared to other buffer.
why I am using DMEM?
I am using DMEM or F12 can also be used as I read in papers that it is used specifically to produce oligomers of abeta 42 full length. People have used DMEM or F12 added into 5mM DMSO dissolved abeta 42 and kept at 4 degrees for 24 hours and observe oligomers by AFM.
Since abeta 25 is equivalent in aggregation and cytotoxicity to abeta 42 full lenth that is why I am using the same conditions to observe oligomers of abeta 25.
Stine, W. B., Jungbauer, L., Yu, C., and LaDu, M. J. (2011) Preparing synthetic Abeta in different aggregation states, Methods in molecular biology 670, 13-32
This paper describes different preps of Abeta42 and might help you.
If the media you use has phenol red in it, that might interfere with the fluorescence readings.
We have used artificial cerebrospinal fluid for protofibrils and fibrils, as described in this paper: Paranjape, G. S., Terrill, S. E., Gouwens, L. K., Ruck, B. M., and Nichols, M. R. (2013) Amyloid-beta(1-42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia, Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 8, 312-322. Perhaps that might work for oligomers.
We have had some difficulties getting Abeta25-35 to form aggregates of any kind, so it is not easy to do. Hope this helps!
thats why I did not have phenol red in it. my DMEM has only glucose. but I have also checked yesterday that when I have only DMEM buffer mixed with ThT there is fluorescence intensity of 150 AU. So i figured out that the buffer which is always kept at 4 degrees and only taken at time of experiment, no matter what has its own fluorescence. but how would have groups like W Klein (Northwestern Uni) etc have done experiments where they obtained abeta 42 oligomers in DMEM or F12. I have checked the above which you have sent already. they have mentioned the same.
Lisa, and also I have tried to get ThT kinetics of the peptide 25-35 in PBS and Tris. They work fine and give a incline and decline kinetics within 6-7 hours in this case i have taken higher conc of abeta say 200ug in one reaction tube for measuring time kinetics. there is a doctoral thesis in 2011 from Texan A&M they also tried to work on this peptide with low conc and achieved kinetics within 24 hours.
Have You used plain DMEM? W/o phenol red and any other stuffs such as serum etcc...
Dmem with glucose only.no phenol red. Mr fabrizio do you gave any idea what could be happening?
Perhaps the other groups have normalized their data to take out the contribution of DMEM fluorescence? If the fluorescence you measured for DMEM alone (150 AU) is subtracted from the Abeta-DMEM measurement in the attached excel data (green curve) then the value is close to the other measurements. I also noticed a red-shift in the Abeta-DMEM peak. Maybe there is something else in the DMEM contributing to fluorescence. There are many types of DMEM available.
dear lisa
i am extremely thankful for your help. I need to figure out what is happening. but my DMEM does not have anything else except glucose. I understand from your paper on modified CSF buffer and abeta protofibrils prep of 42 that F12 can give a pain some of the experiments due to some amino acids added into it. It is also clear that protofibrils grow in this CSF buffer. I can use this one but I need to understand this high fluorescence around 500-550? Please let me know if you happen to see such things in your abeta 42 prep in F12?
Our lab hasn't used F12 since we started using aCSF, and I haven't done ThT for Abeta in F12 or DMEM. Looking at the seller web sites, there are many formulations available, they all seem to have a variety of ingredients in addition to glucose, even without phenol red. Did the manufacturer have any ideas?
Ok. I prepared csf buffer and ran a kinetics with a abeta 21 peptide. Lets see results today. I would like to ask one thing the kinetics for any of these abeta peptide whether full length and shorter fragment is conc dependent?right??. For my abeta 21 which is shortest peptide , i took 160uM for tht kinetics. Frst reading was 106au and within an hour it reached 180AU. So if i hav would taken a low conc like 50uM it would be a different story right??have you taken a higher conc in case of abeta 42 in csf buffer for tht kinetics??pls let me know.
There should be faster aggregation with higher concentrations. Ionic strength, temperature, and shaking can also have an affect. I have not used as high a concentration as you have, I don't recall using more than 40 uM, but I personally have not done much ThT.
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