Hello every one.
i am working on Epsilon15 bacteriophage Purification for EM and cryo EM analysis.
i am using two method to concentrate my bacteriophage (sucrose cushion and 20% PEG-8000 precipitation). after concentrate i run sucrose continuous gradient (30%-70%) to purify the particles as a result i get 3 bands. 1st bands is always empty particles ,2nd band is my desired band that have i need. but third band also looks good in negative staining but particles polymerize.
i need a suggestion how i can increase the 2nd band instead of 3rd band.
Have you tried CsCl density gradient centrifugation ? The high ionic strength may help to dissociate the aggregated phage particles.
For EM analysis, PEG precipitated phage particles are enough. After precipitation you will get highly concentrated phages which is more than enough for EM.
For phage purification, you can use CsCl gradient, that will work good.
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