I do not follow you problema, and not see waht means with exess in your figure, you change the amyloid concentration?

can you maybe try to observed n TEM complete simple, for see other structure formed,  http://www.ncbi.nlm.nih.gov/pubmed/18723507

http://www.nature.com/nprot/journal/v5/n6/abs/nprot.2010.72.HTML

I'm not gonna say what the treatments are because the data is unpublished but it is largely just an inhibitory effect. Also TEM is not useful for me I get more than enough resolution (~0.5nm in air) out of the AFM  I use to observe possible polymorphs, and TEM doesn't allow for in solution imaging which I want to use for this study. I edited my question some to make it more clear hopefully it is a little more understandable now.   

Send me a mail, I do it a lot of aggretation protocol and inhibition with several molecules and maybe I need practic my inglish for follow your problem. I am Agrre with AFM yo can details several details, but in eample you loose macromolecules formation (taurin and ethanol induce abberatn agretation and "pseduplaque" in vitro)

If I can help you... write me

I always dissolve lyophlized A-beta (40 and 42aa long) in 7M Gua-HCl, inject on a SEC column and pick out the monomer fraction. This gives very reproducible results since you always start from the monomer. I attached two articles that describe the method in more detail. I hope this helps you getting reproducable data.

Article A facile method for expression and purification of the Alzhe...

Article Amyloid β-Protein Aggregation Produces Highly Reproducible K...

Please see attached. As you already have found, tremendous differences exist in the way people prepare their Abeta. These are just some additional ideas. Good luck.

Article Preparation of Amyloid β-Protein for Structural and Functional Studies

Article An improved method of preparing the amyloid β-protein for fi...

Thank you very much Dr. Teplow. These papers were a big help with my ThT assays, especially the paper with the ATR-FTIR and CD data.

May be this paper will be useful.

Good luck !

GWAS of longitudinal amyloid accumulation on 18F-florbetapir PET in Alzheimer’s disease implicates microglial activation gene IL1RAP

Vijay K Ramanan, Shannon L. Risacher, Kwangsik Nho, Sungeun Kim, Li Shen, Brenna C. McDonald, Karmen K. Yoder, Gary D. Hutchins, John D. West, Eileen F. Tallman, Sujuan Gao, Tatiana M. Foroud, Martin R. Farlow, Philip L. De Jager, David A. Bennett, Paul S. Aisen, Ronald C. Petersen, Clifford R. Jack Jr., Arthur W. Toga, Robert C. Green, William J. Jagust, Michael W. Weiner, Andrew J. Saykin, DOI: http://dx.doi.org/10.1093/brain/awv231 3076-3088 First published online: 12 August 2015

Vijay K Ramanan

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Shannon L. Risacher

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Kwangsik Nho

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA5 Centre for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Sungeun Kim

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA5 Centre for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Li Shen

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA5 Centre for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Brenna C. McDonald

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA6 Department of Neurology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Karmen K. Yoder

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Gary D. Hutchins

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA

John D. West

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Eileen F. Tallman

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Sujuan Gao

4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA7 Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Tatiana M. Foroud

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA5 Centre for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Martin R. Farlow

4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA6 Department of Neurology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Philip L. De Jager

8 Program in Translational NeuroPsychiatric Genomics, Institute for the Neurosciences, Brigham and Women’s Hospital, Boston, MA 02115, USA9 Departments of Neurology and Psychiatry, Harvard Medical School, Boston, MA 02115, USA10 Program in Medical and Population Genetics, Broad Institute, Cambridge, MA 02142, USA

David A. Bennett

11 Rush Alzheimer’s Disease Centre, Rush University Medical Centre, Chicago, IL 60612, USA

Paul S. Aisen

12 University of Southern California Alzheimer's Therapeutic Research Institute, San Diego, CA 92121, USA

Ronald C. Petersen

13 Department of Neurology, Mayo Clinic Minnesota, Rochester, MN 55905, USA

Clifford R. Jack Jr.

14 Department of Radiology, Mayo Clinic Minnesota, Rochester, MN 55905, USA

Arthur W. Toga

15 Laboratory of NeuroImaging, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Robert C. Green

16 Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA

William J. Jagust

17 Department of Neurology, University of California, Berkeley, CA 94720, USA

Michael W. Weiner

18 Departments of Radiology, Medicine, and Psychiatry, University of California-San Francisco, San Francisco, CA 94143, USA19 Department of Veterans Affairs Medical Centre, San Francisco, CA 94121, USA

Andrew J. Saykin

1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA1 Centre for Neuroimaging, Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Indiana Alzheimer Disease Centre, Indiana University School of Medicine, Indianapolis, IN 46202, USA5 Centre for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA

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Summary

Brain amyloid deposition is thought to be a seminal event in Alzheimer’s disease. To identify genes influencing Alzheimer’s disease pathogenesis, we performed a genome-wide association study of longitudinal change in brain amyloid burden measured by 18F-florbetapir PET. A novel association with higher rates of amyloid accumulation independent from APOE (apolipoprotein E) ε4 status was identified in IL1RAP (interleukin-1 receptor accessory protein; rs12053868-G; P = 1.38 × 10−9) and was validated by deep sequencing. IL1RAP rs12053868-G carriers were more likely to progress from mild cognitive impairment to Alzheimer’s disease and exhibited greater longitudinal temporal cortex atrophy on MRI. In independent cohorts rs12053868-G was associated with accelerated cognitive decline and lower cortical 11C-PBR28 PET signal, a marker of microglial activation. These results suggest a crucial role of activated microglia in limiting amyloid accumulation and nominate the IL-1/IL1RAP pathway as a potential target for modulating this process.

• "final peptide concentration of 20µM"

20 uM is near the critical micelle concentration of ~17 uM for ABeta.

Evidence of the Existence of Micelles in the Fibrillogenesis of β-Amyloid Peptide

http://pubs.acs.org/doi/full/10.1021/jp050716m

The actual CMC varies somewhat depending on conditions. Near the CMC there is an increase in variability in the kinetics (see for example 1B in the "Probing" paper below). Like IAPP, the mechanism of ABeta can also shift depending on the concentration -see:

Conformational Differences between Two Amyloid β Oligomers of Similar Size and Dissimilar Toxicity

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397903/

If you can, it may be better to use a lower concentration far away from the CMC. We have found 5 uM gives good results. By the way, Abeta has another phase transition near 100 uM.

• The signal to noise problem is probably due to differences in the thickness of the fibers. Dense fiber networks as in the top picture  have a tendency to sink in 96 well plates which gives a low detected signal since much of the peptide is no longer in the optical focus. Shaking helps with this but does not completely solve it as the shaking stops during reading.

http://pubs.acs.org/doi/abs/10.1021/jp511758w

check this, if you haven't already see it. It is old but very nice

http://www.ncbi.nlm.nih.gov/pubmed/15937275

I actually solved this issue already [cite: link below]. This is turned out to be an issue with the shaking mechanism of the plate reader. I now have batch to batch reproducible and amazing ThT assay's now. Thank for your help everyone, and Jeffery thanks for the information that's something I'll defiantly check into.

http://www.ncbi.nlm.nih.gov/pubmed/20149780