I would like to isolate amyloidogenic oligomers from the body fluid such as CSF.
Has anyone been successful in isolating oligomers using oligomer-specific antibodies?
Hi Jay, amyloids are specific protein aggregates and I don't know of any chromatographic techniques that can specifically isolate them.
You should post this question to a group on RG that Jeffrey R Brender started and is devoted to sharing amyloid protocols. This group has a number of specialists that study amyloid proteins.
https://www.researchgate.net/post/Amyloid_protocols_project
Dear Jay,
I would suggest you to use asymmetric flow field fractionation, where you don't have any matrix which would otherwise interact with your sample.
You can see how Silveira et al have used this technique in their this elegant study:
http://www.nature.com/nature/journal/v437/n7056/abs/nature03989.html
Best,
Jogender
Hi Jogender, can you point out open access papers that describe this technique?
Hi Steingrimur,
I could not really find any good open access paper on "asymmetric flow field flow fractionation". But here is a link to a nice review on this technique:
http://www.sciencedirect.com/science/article/pii/S0076687906120029
I hope it will be accessible to you. If not, please let me know.
Regards,
Jogender
Hi Jogender, unfortunately I cant access this review.
However, it would be great if you could write up a brief description of this technique and post it a group on RG that Jeffrey R Brender started and is devoted to sharing amyloid protocols.
https://www.researchgate.net/post/Amyloid_protocols_project
Thanks.
Dear Jay
I addressed this question some time ago by using specific anti-oligomer immunoaffinity columns (immuno-precipitation assays). For such task, A11 and Nu1 anti-oligomer specific antibodies were incubated with protein-G Sepharose to create the immunoaffinity column that then was incubated overninght with the sample (at 4ºC). In my case, the sample was soluble protein fraction obtained from tg mouse brain. The sepharose beads were precipitated by centrifugation and mixed with loading buffer for western-blot (SDS-PAGE). It is supposed that SDS-PAGE is not an appropriate technique to analyze Abeta oligomers but it was useful in that case (http://www.ncbi.nlm.nih.gov/pubmed/21460223). I guess you can analyze immuno-precipitated oligomers by other techniques such as native gels or size exclusion cromatogrpahy (described in the literature). Nevertheless, the immunoprecipitation of Abeta oligomers using specific antibodies was a useful technique for our work and it could work in your hands as well.
Good luck!
Dear All,
I am extremely glad to see many valuable information and suggestions of my problem. Thank you all for sharing your opinions.
Best wishes,
Jay
- Badges
- Science method