I am currently conducting research on purifying Myoviridae phage particles using sucrose density gradient ultracentrifugation. However, I have encountered a challenge where no visible opalescent band forms after ultracentrifugation.
I followed the protocol outlined in the paper "Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti‑staphylococcal therapeutic cocktails" (https://www.nature.com/articles/s41598-020-75637-x):
Do you have any suggestions for optimizing the protocol?
Or could the problem be related to the phage titre?
Is it necessary to observe the opalescent band?
Thank you in advance for your assistance.
It is hard to diagnose a negative result, so I can't tell whether you created the gradients correctly etc. But the most likely problem is that you didn't have enough phage. The title needs to be quite high in order to see visible opalescent band, although it depends on the specific phage you should probably aim for 1012 total phage to load (1 liter at 109 PFU/ml for example)
Michael J. Benedik Thank you for your insights. I agree with your perspective; the issue likely stems from the low phage titer. With my titer at only 5 x 10^8 PFU/ml, and considering that I only loaded 2ml of phage into a 13.2 ml ultracentrifuge tube, would you mind sharing a protocol to increase the phage titer? Additionally, I'd like to inquire about the necessity of conducting sucrose density gradient ultracentrifugation for phage purification. Thanks again for your response.
There are two options to increase the phage titer. First would be to optimize the lysis conditions perhaps adjusting the MOI and/or the starting density before adding the phage. Second is to scale up the lysate so instead of 2ml you have 500 ml or even more. You can then collect the phage by PEG precipitation and concentrate it.
Whether or not a density gradient is necessary depends entirely on what you intend to do with the purified phage. If you are just doing simple biological experiments on the phage then there is no need. If you are doing animal studies and such in which case residue from the lysed cell might be problematic, then you may need some purification steps of which a density gradient is one good one (but there may be other options). It might even be enough to PEG precipate the phage.
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