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Author: Subject: nematocysts venom extraction
thechemlife
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cool.gif posted on 14-12-2011 at 19:50
nematocysts venom extraction


I've always had a bit of an obsession with jellyfish and have wanted to investigate them on a more basic level than just looking at the underwater. My idea is to collect specimen (dead of the beach) remove the tentacles and then extract the venom from them to see if it has any interesting properties and see how different species venom functions. Problem is I have no idea how to extract the venom from the nematocysts. I've heard that crushing them in a ball mill releases the toxin which can be collected but am at a lose if this is true and what sort of solvent would be able to isolate the toxin.
Frankly at this point it is just a crazy idea but am looking for some insight on how to do it. If I can't run any decent tests then it'll just be an interesting sample to have in my collection of weird chemicals and materials.
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fledarmus
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[*] posted on 15-12-2011 at 06:25


This might be the preparation you are looking for...

Ramasamy et al, Toxicology Letters (2005), 219


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thechemlife
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[*] posted on 15-12-2011 at 06:51


The article seems perfect but I can't read the part on the extraction. All I can see is the abstract. Does any one have a PDF copy or a way to find the procedure elsewhere?
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Paddywhacker
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[*] posted on 15-12-2011 at 20:56


Here

Attachment: Ramasamy et al., Toxocology Letters (2005), 219.pdf (118kB)
This file has been downloaded 281 times

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thechemlife
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[*] posted on 15-12-2011 at 21:13


Thanks!
ok so from what I gather from the article, the tentacles are kept in seawater to keep them fresh. Then they are removed and loaded into a mini ball mill of sorts. Basically just a screw on top vial filled with small glass beads. This should rupture the nematocysts and release the venom. When enough is collected it is put in a centrifuge tub to remove the left over cell tissue from the venom.

[Edited on 16-12-2011 by thechemlife]
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fledarmus
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[*] posted on 16-12-2011 at 04:52


...and then the material is assayed for activity. Not purity.

I hadn't read far enough down in the article before I posted it to find that their "pure venom" was actually a mixture of at least 12 different proteins, and that previous researchers had found difficulties in separating the proteins due to the instability of the purified proteins. It has also been difficult to tease out the biological effects due to the fact that the mixture of proteins could be affecting multiple pathways.

It looks like an interesting area of study - let me know what you find!
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White Yeti
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[*] posted on 16-12-2011 at 13:09


I think that you would get poor results because jellyfish also have enzymes that are designed destroy the toxins produced after it dies. Complex cytotoxins are usually biodegraded rapidly (anatoxin A for example). You could give it a shot, but most of the toxins are proteins that denature very easily, even upon exposure to light.

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thechemlife
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[*] posted on 16-12-2011 at 13:57


Ya i've been reading a bit more into them and dont think the toxins would make it from collection in florida to extraction in canada. They normally freeze dry the tentacles until they are needed but i'll have no equipment so it wont really be a possibility. I guess it'll be a while till i can try this unless I find a way to get jellyfish shipped for cheap.

Also ya I know it took to long :P I've been using these forums to come up with ideas for a while now, just never had anything interesting to post.
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watson.fawkes
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[*] posted on 16-12-2011 at 15:30


Quote: Originally posted by fledarmus  
...and then the material is assayed for activity. Not purity.
I seems that assaying for activity requires a live animal model, presumably a mouse. Is there another way of assaying for activity?
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thechemlife
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[*] posted on 16-12-2011 at 19:09


Normally they use crayfish as the live model not mice.
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phlogiston
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[*] posted on 17-12-2011 at 14:25


This method is very unlikely to produce any sort of pure venom. It is an extract of the nematocysts, and it will contain thousands of proteins in varying concentration used by the cells in that tissue. The 12 bands shown in the SDS-Page are just the most abundant ones and it is unknown whether these are even part of the venom or just some other abundant proteins from that tissue.

BTW for this purpose the 'ball mill' may not be the best method. This method produces quite a bit of heat, and I find that it sometimes inactivates enzymes I need to extract from tissue. A simpler method that often gives better results (ie more active enzyme) is to freeze the tissue well (preferably with liquid N2), and then grind it up with an equally cold mortar and pestle. Then, suspend the ground tissue in cold (0 ... 4 deg C) buffer, allow it some time to dissolve the proteins (perhaps 20 minutes or so) and finally centrifuge to remove cell debris.
An even better method is to use a potter-elvehjem homogeniser, but you probably don't have access to one.





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