We're collecting tissue at an outside location so we can't immediately snap freeze with liquid nitrogen. One option is to keep the tissue in sterile saline until we can get it back to the lab and snap freeze there. This would involve at the minimum a 15 minute drive. Would this affect protein degradation?
The other option is to freeze on site using dry ice, but I can't find much on whether this is considered appropriate for samples used in westerns.
Freezing intact tissue samples in the field outside the lab is perfectly fine, and you don't really need ethanol. Compared to mRNA, proteins are quite stable, and it will only take 1-2 minutes longer to freeze if you just have dry ice compared to dry-ice ethanol mix. The type of sample and its size is relevant too. Proteases in vacuoles/lysosomes only come in contact with the protein of interest when the cells are damaged (cut, squeezed, crushed...). If you remove small tissue samples by clean cuts, the majority of the cells are undamaged and you can just put them in Eppendorf tubes and freeze them in dry ice. If the samples are large (several grams) and don't fit in Eppendorf tubes, then it might not be good to freeze them at all because you end up with a big solid lump that is difficult to homogenize afterwards, and slow thawing is certainly not good for protein samples because after freezing all the cells are damaged and proteases can come in contact with the protein of interest. You may consider standard ice at 0 degrees Celsius, large boxes and ice-cold sterile saline and drive to the lab. Intact tissue samples kept on ice for 15-30 minutes should be perfectly fine. Whatever the case, the biggest risk of protein degradation is not during tissue sampling and transport, but AFTER the homogenisation step. I would always be more worried about protein degradation during the centrifugation step, and whilst you measure protein concentrations with BioRad and the extracts sitting on ice waiting to be loaded on gel. Good luck :)
Hi, one time i couldn't use liquid nitrogen so as a second option we inverse the samples in isophentane ( -20ªC) that allows you frozen the sample inmeadiatly, you don't reach the tempeture as with N2 liquid but it is an option definitely better that keep samples to room temperature. I don't suggest you put directly samples with ice because you could provide pollution to your sample and probably allows degradation. So may you could use ependorf with isophentane plus dry ice and transport them.
Wouldn't you still need LN2 to get the isopentane to -20? It does not maintain the temperature by itself. At least that's how we do it. I have frozen samples on dry ice (not directly but in 0.5 ml eppendorf tubes) for both RNA and protein and have not had any degradation.
In our lab we usually carry liquid nitrogen with thermical coffee/tee bottles. So in the collection area, we put samples into 2 mL eppendorff tubes and let them into the bottle. A full filled bottle holds LN2 for about 8 hours.
PS. Never lock the bottle completely or else the bottle gets pressure. Instead, just loose a little.
I guess you could snap freeze your samples in eppendorf tubes using an ethanol bath with dry ice, then transport them in dry ice.
Freezing intact tissue samples in the field outside the lab is perfectly fine, and you don't really need ethanol. Compared to mRNA, proteins are quite stable, and it will only take 1-2 minutes longer to freeze if you just have dry ice compared to dry-ice ethanol mix. The type of sample and its size is relevant too. Proteases in vacuoles/lysosomes only come in contact with the protein of interest when the cells are damaged (cut, squeezed, crushed...). If you remove small tissue samples by clean cuts, the majority of the cells are undamaged and you can just put them in Eppendorf tubes and freeze them in dry ice. If the samples are large (several grams) and don't fit in Eppendorf tubes, then it might not be good to freeze them at all because you end up with a big solid lump that is difficult to homogenize afterwards, and slow thawing is certainly not good for protein samples because after freezing all the cells are damaged and proteases can come in contact with the protein of interest. You may consider standard ice at 0 degrees Celsius, large boxes and ice-cold sterile saline and drive to the lab. Intact tissue samples kept on ice for 15-30 minutes should be perfectly fine. Whatever the case, the biggest risk of protein degradation is not during tissue sampling and transport, but AFTER the homogenisation step. I would always be more worried about protein degradation during the centrifugation step, and whilst you measure protein concentrations with BioRad and the extracts sitting on ice waiting to be loaded on gel. Good luck :)
I would be careful in using dry ice as dry ice is hygroscopic in nature and it is home for bacterial contaminations. Enough analysis is done to prove that dry ice can not be used for sterile environment.
If you have indirect cooling then it may be okay, but for biological samples, if it is direct contact, I will not use dry ice. LIN ( liquid nitrogen) may be an alternative or other indirect cryogen. But, to avoid shock of extreme temperatures on your samples, when you use liquid nitrogen, you want to have indirect cooling using cooling media. Hope this helps.
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