2 replies to this topic

[fishdoc]

I'm looking for a protocol for purification of proteins from macrophages infected with a facultative intracellular bacterium. I do not want to collect bacterial proteins, however. I have FLAG-tagged a number of bacterial proteins that I suspect to be secreted, and want to detect them from macrophage lysates using western blot. I'm not sure if the proteins are targeted to the cytoplasm or the nucleus (or elsewhere?). I've found a number of methods for lysis of cells in culture, but I'm unsure if most of them will also result in the lysis of the bacteria. I need a protocol that ensures the bacteria are kept intact, so that I can be sure the detected proteins were secreted.

We do a gentamicin exclusion assay using 0.1% triton x-100 to lyse the cells, and the bacteria remain intact. Would it work to lyse the infected macs with that, collect the lysate, pellet the debris (and bacteria), and purify the protein from the supernatant, or would the triton x-100 interfere somehow?

Any suggestions or comments you could give are greatly appreciated.


Edit: I was mistaken, the triton x-100 concentration is 1%, not 0.1%.

Edited by fishdoc, 11 December 2009 - 08:12 AM.

[Go_johnny_Go_Go_Go_Go]

I do infection of macrophages with mycobacteria (pretty hardy bugs)

i lyse the cells using m-per from pierce (plus protease/phosphatase inhibitors), then spin out the bugs and ceullar debris. the lysate then gets analysed by western for signalling molecules..

m-per would be compatible with your downstream applications,
i suspect the triton protocol would be ok also as its only 0.1%, but it would depend on your purication conditions

[fishdoc]

Go_johnny_Go_Go_Go_Go, on Dec 11 2009, 09:59 AM, said:

I do infection of macrophages with mycobacteria (pretty hardy bugs)

i lyse the cells using m-per from pierce (plus protease/phosphatase inhibitors), then spin out the bugs and ceullar debris. the lysate then gets analysed by western for signalling molecules..

m-per would be compatible with your downstream applications,
i suspect the triton protocol would be ok also as its only 0.1%, but it would depend on your purication conditions

I had looked at m-per as a possibility for this, so that is good to know. I was mistaken above when I wrote 0.1% as the triton concentration for lysis, it is 1%.

My initial thoughts were that I could either run lysate directly on a gel, or TCA precipitate it, but I wasn't sure what the effect of triton would have on either of those methods, if any.

I think I may order a small volume of m-per and see what it does to a bacterial pellet... if it doesn't lyse the pellet, I'm good.