Hi,
do you know a protocol for the extraction of outer membrane proteins from Gram negative bacteria? I am working on Klebsiella pneumonia and I don't have a sonicator avalabale in my lab?
Thanks,
Silvia
Sorry I realized that I don't have an ultracentrifuge either in my lab. How can I do? Are there kits for this?
Thanks again
Dear Silvia. You can manage without sonicator (there are many other methods like French press, detergents, etc.) but you have use the centrifuge after all. As far as I know, there are no kits to isolated outer membrane proteins.
You can lyse cells without sonicator, but there is no way of purifying native (not tagged) OMPs without using an ultracentrifuge. The closest you can get is to isolate outer membrane vesicles (OMVs), which can be done by filtration of cell-free supernatant. However, the OMV are enriched in outer membrane proteins but also contain periplasmic proteins and this approach won't give you the whole picture of the outer membrane proteome as some OMPs are not well represented in OMVs.
Dear Silvia,
Here am sharing regarding outer membrane research article from BIO-RAD.
First of all have a look the document you will get a brief idea.
Please go through this PDF
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2057.pdf
You can extract membrane protein by lysing the cells with chemicals. and separating the supernatent containg the membrane proteins
Hi Silvia,
I had extracted OMP of Bordetella pertussis With Brij 35, a detergent. You can employ a heat extraction if your protein of interest can withstand a level of temperature.
In my case I had extracted Pertactin protein that can withstand 80°C. If you protein can not withstand the heat then you can use sterile inert beads to disrupt the bacterial cells and then centrifuge to take the supernantant and then purify it.
In this paper you will find several methods for lisis/fractionation.
Efficient subfractionation of gram-negative bacteria for proteomics studies.
J Proteome Res. 2010 Dec 3;9(12):6135-47
Cheers!
If your protein is not toxic to the organism you are expressing it in, then you can try using pCold vector for expression. it slows down translation of other proteins, while that of the protein's gene inserted in the vector will express a lot more. Read this pdf that I have attached with this message once, I don't think you would need a lengthy protocol for its extraction either:
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