My posting has been a bit infrequent because I've been focused on getting drafts of our two grant proposals ready for pre-submission critiquing by the volunteer reviewers (blessed be their names and their progeny). But that's done, with a lot of help from a grad student and a postdoc. I've committed myself to the Week of Science Blogging, so I'll be posting at least daily for the next week.
We put a fair bit of effort into crafting a tightly focused "Significance" section to end each proposal (after the detailed descriptions of the experiments we propose). It's important to avoid ending with strings of platitudes, so ours is a bit on the edgy side. On the other hand we can't afford to sound too arrogant (this is a Canadian granting agency).
Some science: We had been hoping to be able to use preparations of cell membranes to study how a radioactively-labeled 'test' DNA fragment interacts with the uptake machinery. But we had overlooked the problem of all the cellular DNA that will be released when we break the cells open to get the membranes.
When the cells are first opened the uptake proteins will bind the abundant cellular DNA, and unless we get rid of the DNA they've bound, they will never bind our test DNA. In principle we could get rid of the cellular DNA by adding a lot of DNase I, an enzyme that breaks down DNA. But then we would need to get rid of ALL of the DNase I , as otherwise it will break down our test DNA!
The postdoc (who discovered the problem) is going to do some preliminary testing to see if this analysis can be salvaged, perhaps by repeatedly washing the membranes to get rid of the DNase I.
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ReplyDeleteCan you do your assay with intact cells? Then you could treat the outside of the cells with DNaseI followed by EDTA/proteinaseK, as we did with intact HIV virions (see methods here). Virions are not lysed by the protK, so they efficiently protect viral DNA from the DNaseI, which in turn is efficiently destroyed by the protK. I don't see why prot K would lyse cells, which would protect any labeled DNA they had taken up. You can either boil or phenol/chloroform extract after protK treatment; I suggest trying both because my replacement in that lab found that the two methods of protK removal gave different results.
ReplyDeleteHi Bill,
ReplyDeleteYes, intact cells are our fall-back. But the density of uptake machinery per cell is seriously limiting for the kinds of analyses we are hoping to do. We were hoping that using membrane preps would enrich for uptake machinery, eliminating a lot of the irrelevant background material.