I used E coli to infect my cell (J774 macrophage) and want examine the proteome of J774 after infection by shotgun proteomics. Though I can filter the spectra corresponding for the E Coli using Mascot, are there any method to separate them?
Culture the cells on Mac Conkoy medium the bacteria will grow on the media and you can separate them while the other cells will die
how about short time or mild tripsinisation and washing sevral times with PBS along with centrifugation at low rpm (1500) ?
Thanks for your suggestion. Just in case, are there any method to separate the bacteria in the cell from the protein of the cell? Thanks.
I think if you transmit your cells and put them on solid selective bacterial media like Mac Conlkey agar media the macrophage will die and dissentegrate and the. E colia will over growth. And you can make pure subculture on another plate
If you wante to separate the proteins of the cells you can dissentegrate the cells the put the suspension to pass through bacterial filter the protein of the cell will pass through the membrane while the bacteria are trapped. Then take the filtered fluid and centrifuge it. The deposited will be the cell priotein
How fast has the isoltaion to be? Do you expect changes in the proteome? Wath is your time Frame?. You could first positive isolate the cells- wash the free bateria away and then break up the cells and ioslate your bacteria by growing on agar or by positve isolation in case you know suiltable antibodies.
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