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Fantastic-Climate-84 t1_j814pfj wrote

Reply to comment by Natolx in Can the Radiation from a Sample of Depleted Uranium Sterilize? by Natolx

First, I love this post and this is why I follow the sub.

Second, are you performing any experiments that have been, potentially, been contaminated?

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Natolx OP t1_j81a45e wrote

Essentially I made a piece of jewelry containing a concentrated solution of fluorescent protein. It began as a sterile solution and a sanitized glass ampule, but a mishap during the final step sealing it up (with me breathing over top) may have unfortunately introduced some contamination.

I didn't include any toxic preservatives like sodium azide for safety reasons in case it ever breaks.

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RebelWithoutAClue t1_j81awnc wrote

It may be that a thorough irradiation sterilization will end up denaturing your protein anyways.

Maybe one could accept that a beautiful thing could be beautiful also because it is temporary.

At what temp will your protein denature? If the protein denatures at a high enough temp, you could hold it at a lower temp to make life really hard for bacteria and wait it out for a few days to kill it down while not denaturing your protein.

Basically pasteurize the thing at a temp that is below the temp that will damage the protein you care about.

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Natolx OP t1_j81cnjx wrote

No worries about the denaturing, fluorescent proteins are notoriously stable structures. They are even resistant to Proteinase K... which is ridiculous.

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[deleted] t1_j81maix wrote

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Natolx OP t1_j81vyay wrote

>Without being replenished by new protein, the GFP would photobleach pretty quickly anyways, so I don't think the necklace is worth trying to sterilize. While GFP is stable to protease digestion, it doesn't really remain fluorescent in a purified solution for more than a few days if you just leave it our like you would with normal jewelry. Plus, killing the bacteria probably won't improve the appearance, as they likely broke down a lot of your GFP for food and their corpses will keep making the solution cloudy even if they're no longer alive. Honestly, it's probably easier to write this one off and make a new one.

I think you underestimate how much fluorescent protein we are talking about... This is milligrams. This is many orders of magnitude more protein than you are seeing photobleached in an immunofluorescence assay.

To put this in perspective, this liquid containing fluorescent protein entirely absorbs a 473nm laser I have. None of it makes it out the other side.

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Camilo543 t1_j81z2fz wrote

Would this make UV sterilization impossible?

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Tiny_Rat t1_j822bd1 wrote

UV sterilization would definitely photobleach GFP in the time it took to kill the bacteria.

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Natolx OP t1_j822zcg wrote

>Would this make UV sterilization impossible?

Yes, unfortunately the fluorescent protein would absorb the UV and protect the bacteria! Although there is a small chance UVC might work.... That is well outside of the absorption spectra of the protein

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[deleted] t1_j820ohg wrote

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Natolx OP t1_j822m5l wrote

> Regardless, even large quantities of purified GFP need to be stored at -20C in the dark to remain fluorescent long-term, and raising that temperature to even 4C will dramatically shorten storage time. The rate of photobleaching and degradation at RT isn't going to be affected by the concentration of the protein, it just might take slightly longer to see a difference by eye. This is still a very temporary piece of jewelry, so sterilizing it at this point isn't going to extend its lifespan by much.

I'm sorry but this is simply incorrect. Photobleaching is by definition caused by light being absorbed. A high concentration of protein on the "shell" of the solution is going to "protect' all of the protein on the inside of that shell from from excitatory light. Again, I don't think you can conceptualize how much protein this is compared to "normal" amounts seen in laboratory experiments.

If I left it out in the sun, sure, it's going to bleach for in a week, but the photobleaching power of incidental lighting is just not enough to photobleach this amount of fluorescent protein any time soon.

I have tubes of nonsterile fluorescent protein that have been kept at room temperature for a year now that are cloudy (with contamination) but still fluorescent. Only my sample kept in the sun lost fluorescence.

Additional Note: this is mNeon, not GFP so it is definitely a "better" generation of fluorescent protein. But even GFP at this concentration is going to resist photobleaching for an absurdly long time.

>The "absorbtion" you see with your laser beam probably has more to do with scattering of the laser rather than pure absorption. Any high-density protein solution will behave similarly.

There is no blue light being "scattered" (I have used a blue filter I scavenged to check) , it is not scattering. You can also clearly see the beam go in, stay a beam but just fade into nothing.

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EmilyU1F984 t1_j81ib1n wrote

Why not just heat sterilize it? If you do that in a pressure vessel the pressure inside your jewelry and surrounding it will be the same, and it thus won’t explode.

Also is there anything in there but the protein? Even if you introduced something, stuff won‘t be able to grow on just a single protease resistant protein snywsy.

Not like it‘s meant to be used for injection. Just needs to not go opaque right?

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Natolx OP t1_j81w5ly wrote

Nothing but protein, but there is phosphate from the buffer as well.

Edit: An autoclave would almost certainly denature it. There is resistant to denaturating and then there is resistant to autoclave lol. Prions are one of the few proteins that are resistant to autoclaving and they are considered exceptions.

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EmilyU1F984 t1_j83abzg wrote

Which would depend on your specific protein, and prions aren‘t that specific.

Sure globulins will be gone and other unstable protein, but since you didn‘t mention the protein.

Can just pasteurise in an autoclave as well.

No need to do 121C if you aren’t going for medical sterility. Most stuff does way earlier.

Well phosphate and protease resistant protein don‘t make up a very good growth mediums

So not much risk of colonies forming anyway.

Though got any further attempts you got stuff like propylenglykol or regular preservatives available. No use to go toxic.

Could even just use thiomersal if you still got some lying around.

But sorbic acid if acidic or parabenes if neutral to whatever if basic. That stuff works for creams that people touch daily just fine to prevent growth.

Other way round, find someone with an x ray in a lab and just use that.

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Tiny_Rat t1_j81m6ud wrote

If they autoclave it, a sealed vessel will still explode, as the pressure inside the vessel and the machine will still change at different rates. Plus, killing the bacteria won't make the solution less cloudy, it will just be cloudy with dead bacteria instead of living ones. And, worst of all, heat treatment will most likely kill the fluorescent proteins anyways.

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RebelWithoutAClue t1_j82asnn wrote

It could be autoclaved in a pressure cooker if the protein can tolerate the temp.

In a pressure cooker the pressure is also exerted on the outside of the ampule. No pressure differential, no kaboom.

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