Please some one suggest me a good protocol, giving high yield of ER membrane and nuclear protein (if both protein extraction in same buffer, then better) from agroinfilterated Nicotiana benthamiana leaves. I want to do western blott and my proteins are ER membrane and Nucleus located with low concenteration.
Thanks.
Please see attached.
This is a protocol for the isolation of total microsomes and plasma membrane. Please disregard the section on the plasma membrane. The "total microsome" fraction is ~35% ER, 35% chloroplast thylakoids, 20% Golgi, 10% vacuole, and 10% plasma membrane. While this is not a high purity endoplasmic reticulum prep, it is a high yield prep.
thank you so much for the protocol.
though i have a question, as in my sample I am extracting both ER membrane protein as well as nuclei protein. in above mentioned protocol there is no protein extraction from nuclei.
let me clear more about my experiment: I am doing agroinfiltration of GFP tagged my protein. then these leaves were treated for 24 hrs. Now i want to do protein immunoblot in which my protein is located in ER but after some treatment it is localized in Nucleus. So i want to extract ER mem protein in untreated samples and in treated samples I need expression of botth ER membrane and nuclei protein.
thanks
It is easier to answer your question by fluorescence microscopy. If you want to see an increase in the nucleoplasm, and a decrease in the nuclear envelope and endoplasmic reticulum, then focus on a nucleus of an expressing cell and do a line-scan through the centre of the nucleus (pinhole should be no more than 1 micron). You do that for 20 nuclei of "untreated" leaf epidermis cells, and 20 nuclei from "treated" cells. The nuclear envelope and endoplasmic reticulum are connected, and there is a good correlation between the fluorescence in these two compartments when the protein resides in the ER (lumen or membrane). If the protein re-partitions to the nucleoplasm during your treatment, then your signal at the periphery of the nucleus (the envelope) should decrease and the signal inside should increase. It should be easy to see by microscopy, but to do this by cell fractionation is very difficult, because the ER emanates from the outer nuclear envelope membrane, so everything is connected. However, if you fractionate intact tissues, you are going to break many nuclei (and loose nucleoplasm), and if you make protoplasts first to do gentle cell lysis afterwards, it might affect your biological conditions.
Dear Denecke,
thanks for replying. I did the florescence microscopy and got the signal in ER without treatment and after treatment its relocating in nucleus but to support that data I need Protein data also (immunoblot). kind of mandatory data.
My main concern is concentration of my desired gene, because during microscopy the GFP expression is not too high.
Regards,
Pratima
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