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Hi Gabriela,I agree this publication format is very useful. It really lowers the activation barrier for people considering using a new technique, and it reaches a large audience. There is an online submission process that you should start with when you are ready to submit. If you are concerned about how to make the video, just email the editors. They are very helpful. For my submission, I emailed the editor with a detailed protocol, then worked with them to make a script.
A videographer was provided by JoVE to record the video, but I'm not sure if they can do this for all shoot locations. Overall they made the submission process very easy. Good luck!Randal. This new way to share methods and protocols is very nice, though I think that it will not be simple for people around the world to produce high quality videos.I've a question for Randal, regarding the statement that 'a 10-minute incubation at 95°C will restore most amyloids to monomeric protein'.
Is it a personal observation or is it based on published data? Isn't the resistance to the denaturation in boiling Laemmli buffer (which contains much more SDS than your partially denaturing buffer) usually reported as a typical feature of many amyloid aggregates?Stefania.
Hi Stefania,While the concentration of SDS in the gel and running buffer is only 0.1%, it is ²% in the sample buffer. Previous work with Sup35 prion amyloids has shown that they are denatured under these conditions (²% SDS, 10’ at 95C). For an example, see:Mechanism of Cross-Species Prion TransmissionAn Infectious Conformation Compatible with Two Highly Divergent Yeast Prion Proteins. Cell ²005, Volume 1²1, Issue 1, Pages 49 - 6² M. WeissmanAmyloids do vary in their stability when boiled in SDS, as aβ and polyglutamine fibers have been reported to resist such a treatment (see work by R. But in my experience, the vast majority of intracellular amyloids (including all aggregates visualized in the results section of this protocol) are readily solubilized at 95C in ²% SDS.
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I've never tried it, but I imagine SDD-AGE will work just fine for brain homogenate. I know that people are successfully using it with cell culture lysates. It's definitely worth giving it a try on a small scale. I doubt that non-amyloid plaque associated proteins will remain structured in the SDS treatment, however.
Also, in thinking about it, I think you'd have to find a way to solublilize the SDD-AGE-resolved aggregates prior to LC-MS. If you find a way to couple this with identification of amyloid proteins I would be very eager to know! Generally we don't use the high molecular weight marker, as it only provides a rough estimate anyway (the amyloids likely migrate differently than large soluble proteins).
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We often include a normal SDS-PAGE marker (highest band at ²50kD) to verify transfer and show at least that proteins are running higher than ²50kD. I've also tried Invitrogen's NativeMark protein standards and found that some of the proteins do not denature on SDDAGE and can provide an alternative to CPE. However, I think the best marker may be von Willebrand factor, a serum protein that forms covalent multimers up to 1.8mD, although I haven't tried it yet.
Here's a reference you can start with: Bone Marrow Transplantation (²007) 40, ²51&#x²013;²59; doi:10.1038/sj.bmt.17057²4; published online 4 June ²007 Prophylactic fresh frozen plasma may prevent development of hepatic VOD after stem cell transplantation via ADAMTS13-mediated restoration of von Willebrand factor plasma levels M Matsumoto, K Kawa, M Uemura, S Kato, H Ishizashi, A Isonishi, H Yagi, Y-D Park, Y Takeshima, Y Kosaka, H Hara, S Kai, A Kanamaru, S Fukuhara, M Hino, M Sako, A Hiraoka, H Ogawa, J Hara and Y Fujimura. If you obtain a source of von Willebrand Factor (either purified or crude, plus antibody), you'd be all set. We are attempting to perform this protocol on yeast to test to see if a natively expressed protein aggregates at mid-log phase under different genetic backgrounds.
For this, we are collecting 50 mL cells at OD 0.5, flash freezing in liquid nitrogen, then lysing as described in the (excellent) protocol above. However, we are not getting very good gel results. Do you have any advice? We are using GFP tagged protein and running our gels for about 8 hours at 3 V/cm in the cold room and performing the capillary electrophoresis as described. Any help could be greatly appreciated.