SEA-PHAGES | Capsid denaturation

Link to this post \| posted 27 Oct, 2021 19:27
stpage	Hi, the phage discovery has RNase/DNase/ProteinaseK treatment followed by CleanupResin (guanidinium thiocyanate) to denature the capsid. Some protocols seem to use proteinaseK to denature the capsid. (https://www.mdpi.com/2409-9279/1/3/27/pdf-vor) and some seem intermediate (https://pubmed.ncbi.nlm.nih.gov/29417429/). Is it very phage dependent? With new hosts do we need to optimize the proteinaseK treatment??

Link to this post | posted 27 Oct, 2021 19:27

Hi, the phage discovery has RNase/DNase/ProteinaseK treatment followed by CleanupResin (guanidinium thiocyanate) to denature the capsid.
Some protocols seem to use proteinaseK to denature the capsid. (https://www.mdpi.com/2409-9279/1/3/27/pdf-vor)
and some seem intermediate (https://pubmed.ncbi.nlm.nih.gov/29417429/).

Is it very phage dependent? With new hosts do we need to optimize the proteinaseK treatment??

Link to this post \| posted 27 Oct, 2021 21:43
viknesh	stpage Hi, the phage discovery has RNase/DNase/ProteinaseK treatment followed by CleanupResin (guanidinium thiocyanate) to denature the capsid. Some protocols seem to use proteinaseK to denature the capsid. (https://www.mdpi.com/2409-9279/1/3/27/pdf-vor) and some seem intermediate (https://pubmed.ncbi.nlm.nih.gov/29417429/). Is it very phage dependent? With new hosts do we need to optimize the proteinaseK treatment?? Hi Shallee, The guanidinium thiocyanate in the cleanup resin is enough to denature phage capsids, at least for all the phages we work with. If you or anyone else is having trouble with DNA isolation, please let us know so we can look into this. As for the additional "optional" ProteinaseK + SDS step in the guide, this was added because we were observing some nuclease activity co-purifying with DNA for some phages isolated on M. foliorum. You'll know if you have this problem if your phage DNA looks good on a gel if the sample hasnt seen any cations (which are needed for DNase activity), but is degraded if it has. For example, even the addition of restricton enzyme buffer (i.e. for the DNA control with no actual restriction enzyme) would result in degradation of phage DNA, but loading the DNA sample directly onto the gel results in the typical nice high-molecular-weight band (with little to no smear). While we dont know why nuclease co-purifies for M. foliorum phages, or even what the nuclease is (whether its the nuclease we add or some M. foliorum nuclease), adding ProteinaseK and SDS after treating lysates with nuclease resolves the problem. As you'll recall, we typically simply inactivate the nucleases by adding EDTA. For those working with M. foliorum, or for those seeing nuclease activity co-purifying with their phage DNA, the addition of Proteinase K and SDS that is able to degrade that nuclease can resolve the problem. I hope this is helpful. Vic

Link to this post | posted 27 Oct, 2021 21:43

viknesh

stpage
Hi, the phage discovery has RNase/DNase/ProteinaseK treatment followed by CleanupResin (guanidinium thiocyanate) to denature the capsid.
Some protocols seem to use proteinaseK to denature the capsid. (https://www.mdpi.com/2409-9279/1/3/27/pdf-vor)
and some seem intermediate (https://pubmed.ncbi.nlm.nih.gov/29417429/).

Is it very phage dependent? With new hosts do we need to optimize the proteinaseK treatment??

Hi Shallee,

The guanidinium thiocyanate in the cleanup resin is enough to denature phage capsids, at least for all the phages we work with. If you or anyone else is having trouble with DNA isolation, please let us know so we can look into this.

As for the additional "optional" ProteinaseK + SDS step in the guide, this was added because we were observing some nuclease activity co-purifying with DNA for some phages isolated on M. foliorum.
You'll know if you have this problem if your phage DNA looks good on a gel if the sample hasnt seen any cations (which are needed for DNase activity), but is degraded if it has. For example, even the addition of restricton enzyme buffer (i.e. for the DNA control with no actual restriction enzyme) would result in degradation of phage DNA, but loading the DNA sample directly onto the gel results in the typical nice high-molecular-weight band (with little to no smear).

While we dont know why nuclease co-purifies for M. foliorum phages, or even what the nuclease is (whether its the nuclease we add or some M. foliorum nuclease), adding ProteinaseK and SDS after treating lysates with nuclease resolves the problem. As you'll recall, we typically simply inactivate the nucleases by adding EDTA. For those working with M. foliorum, or for those seeing nuclease activity co-purifying with their phage DNA, the addition of Proteinase K and SDS that is able to degrade that nuclease can resolve the problem.

I hope this is helpful.

Vic

Link to this post \| posted 27 Oct, 2021 23:34
stpage	Thanks, Vic. That is helpful. We were losing some DNA and weren't sure if we were getting capsid denaturation from the proteinase K. Different protocols seem to have wildly different proteinase K conditions.

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