Dear Fang,
When I get the plant nucleus, I just want to use...
Dear Fang,
When I get the plant nucleus, I just want to use it for western blot to detect certain proteins. Can I use the GTEN buffer (GTEN, 2%w/v PVPP, 10m M DTT, 1×protease inhibitor cocktail, 0.1% Tween 20) to extract the nuclear protein, as we usually use this buffer to extract Nic Read more Read less

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Fang Xu Author Answered Aug 9, 2020
Shandong University
Hi XingJie,
1, I used to centrifuge 10mins for 2 time to get rid other pellet.
2, I think it is not necessary to wash the plasma membrane, you can directly extract proteins.
3, for nuclear and plasma membrane, you can directly add 1X SDS buffer while for cytosol you should add 2X SDS buffer so that the final concentration would be 1X SDS.
Fang
Dear Fang,
Thanks for your reply. Before getting the nuclear protein, I also plan to extract the proteins of cytoplasm and plasma membrane respectively. As the protocol above, the suspension after Step 4 contains plant cytoplasm and plasma membrane. Collecting the supernatant, we can centrifuge at 16 000 g and ultracentrifuge at 100 000 g to get the supernatant for cytoplasm for the plasma membrane. Here are two questions hopefully to be solved.
1. How long it takes to centrifuge at 16 000 g to get rid of other pellets?
2. When I get the plasma membrane pellet, which buffer should be used to wash and resuspended the pellet to get rid of contamination? Is it necessary?
3. Can I use the same 1X SDS buffer (just as your suggestion) or the GTEN buffer to extract the nuclear, cytoplasm and membrane protein respectively?
Bests,
Xingjie
Fang Xu Author Answered Jul 28, 2020
Shandong University
Hi Xingjie,
If you only want to detect the protein after you extracted the nucleus, you can also use 1X SDS buffer to extract the total nuclear protein. In terms of your GTEN buffer, I think PVPP is not necessary and maybe you should also include Tris, some salt (Nacl or Kcl) and glycerol. Hope this helps.
Fang.
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