>Glass breaks when there's a temperature differential between one area and another. > >Heated from room temp in a water bath, it probably won't happen.

OP was talking about freeze thaw. The expanding of the ice would be the problem.

>A few cycles of freeze-thaw alone will kill the bacteria fairly effectively, the problem is that it may break your glass vial and damage the protein inside.

The protein will be fine. It's already been freeze thawed a bunch of times in a plastic tube. But yes, the glass breaking is a problem.

>The gm500 has two tubes right? I don't know how it chooses which one to use (probably changes after it gets saturated), but if the standart tube is something like a sbm-20 or j305 it probably picks up quite a bit of beta that goes through the ampoule, and the cpm to usv is most likely calibrated with the energy of cesium, so i think the actual doserate is lower that what it shows

I placed whichever tube was more sensitive over the sample (one of them barely detected anything). Good call on the calibration.

>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

> 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.

>The dose rate seems to be very high for DU gamma radiation. I calculated 400nSv/h using 0.7 Sv/Gy on contact for 0.2 w/o U235 for newly depleted material. The dose rate will increase a bit as daughter products accumulate over thousands of years, but that isn’t particularly relevant.

This is just the default calculation of cpm to microsieverts by my radiation counter (GM500) so I suppose it could be that the calculation is wrong.

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.

>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.

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

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.

>I think that freezing should allow one to achieve sterilization with a low emission source by basically tilting the replication rate to kill rate in a severe way.

>Low emission rate sources need more time to achieve sterilization.

>Still though, depleted uranium generally provides alpha and beta emissions with a bit of gamma. The alpha won't make it through the walls of the ampule. I think that much of the beta also won't make it through. You'll be dependent on the low gamma emissions to slowly achieve your sterilization.

The 15 microsieverts per hour measurement should already take that into account. The uranium sample itself is in glass so all of the alpha and most of the beta should be contained.

>This is roughly the dose rate of simply being on a plane at cruising altitude.

My initial research suggested this rate was 5 times that of cruising at altitude, but your point is taken. Thank you.

What if the bacteria/fungus in the starting sample were unable to propagate in the liquid? Either no food or maybe even frozen for the duration of the "treatment"?