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Whole tissue lysis vs. Organelle (chloroplast) lysis


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#1 nimbus

nimbus

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Posted 07 February 2013 - 01:41 PM

Hi,

I'm curious about a question regarding lysing plant cells in order to use the lysate for subsequent IP because I just had an argument with my boss about it and no one of us had a real answer to it. So is there a difference in the quality of the lysate (e.g. components that might interfere with the subsequent IP) whether one uses the whole tissue, homogenizes it in lysis buffer and perform the IP with the supernatent or whether one extracts the desired organelle first (chloroplasts in my case using a buffer with an osmoticum e.g. sorbitol), collects the intact chloroplasts by centrifugation and then lysis those intact chloroplasts.
I can see that it makes sense to isolate organelles if you really want to study them but I don't see the purpose of isolating them when you only want to use the lysate for a subsequent IP. Especially since in my case I'm working with a protein that is not very abundant and I loose a lot of when I only take intact chloroplast (upscaling is not a solution because you need A LOT!).
Any opinion welcome! Oh, and btw, both methods work to IP the protein with the difference that the yield after whole tissue lysis is much much more.
Cheers and thanks




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