RIPA and bacteria - Will they lyse? (Jan/21/2010 )

I'm trying to figure out a protocol to use for studying proteins secreted by intracellular bacteria. I want to be able to lyse infected macrophages to harvest the macrophage protein (including any proteins secreted by the bacteria into the macrophage cytoplasm), and I want to be able to show that the protein wasn't simply due to lysed bacteria. The assays will be done in either 6 or 24 well plates using primary cultures of macrophages. My thought was to use RIPA buffer to lyse the macrophages, then after centrifugation of the lysate, sonicate the pellet, which should include bacterial proteins. I am not sure, however, that RIPA will not lyse the bacteria. It's a Gram negative organism. I can't find any definitive answer on this, so was hoping someone here might have a little insight. I plan on growing up a culture of the bacteria tonight, pelleting, and resuspending in PBS or RIPA buffer, then plating to determine if there was any loss of CFUs/ml after exposure to the RIPA. That should tell me if there is any effect, but I had hopes maybe someone here knew for sure so that I could skip having to test the bacteria in the buffer.

I know the bacterium holds up against 1% triton x-100, because that's what we use to lyse the macs in gentamicin exclusion assays, but I'm not sure how it will handle the 1% deoxycholate or the 0.1% SDS. Actually, I'm not too worried about the 0,1% SDS, more the deoxycholate, or the effect that all three detergents could have on the bacteria as opposed to just one of the detergents.

Any info would be appreciated.

Also, if anyone has an established protocol or an abstract describing a protocol to do what I've described above, that would be great, too.

Alright, I did a quick and dirty assay to determine the bacterium's stability in RIPA versus physiological saline. Pelleted 8 x 250 ul of an overnight culture. Resuspended the cells in saline or RIPA buffer (250 ul) in 4 ways: vortex, flick the tube, pipetting, or resuspending first in 50 ul saline by vortex, then adding 200 ul of saline or RIPA. Did these methods to determine if I had to be careful with the cells in RIPA or could be relatively harsh. Incubated the resuspensions at 4 C for 20 min. After incubation, serially diluted each to 10^-8 and drop plated 20 ul of each dilution and grew overnight to determine if there was any appreciable loss of cells due to the RIPA buffer. Counted colonies today, and long story short, the bacteria did not experience any detectable lysis. The calculated CFUs/ml were the same in either treatment, between 1 and 2 x 10^10 CFU/ml.

A side note, however, is that I left the saline and RIPA resuspensions on the benchtop overnight, and all the RIPA ones were pretty clear, whereas the saline ones were still cloudy. I didn't plate those to verify, but I'm pretty sure that the extended incubation at room temp overnight did result in lysis of the bacteria. The protocols I saw for RIPA, though, are incubations up to 20 minutes, and for that time period, the bacteria seem pretty resistant.

Thought this info might be useful for anyone else that may be interested in doing similar experiments with intracellular bacteria.