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TCA precipitation - (Feb/26/2013 )

Hi there,
I have to do a cell wall extraction on bacteria soon and my protein of interest is cell wall anchored. My supervisor told me to do TCA precipitation (haven't done before) but I assume it's too isolate the proteins within the cell wall?? He told me I would have three components by the end of it, the soluble, insoluble and something else!!! (the isolated proteins??) arrg I have looked at a protocol for a cell wall extraction. Here is a simplified version below

Wait till OD 0.6
Spin at 5000rpm for 10 mins
Remove Supernatant and resuspend pellet in PBS
Spin at 5000rpm for ten mins
Discard Supernatant and resuspend pellet in 1 mL protoplast buffer. Incubate for 3hours at 37C
spin at 13K for 15mins
store supernatant at -20C

So the supernatant will contain cell wall and the insoluble protoplast component will be left right? So is it the supernatant at the last step i TCA precipitate? if it is, i've looked at a protocol for TCA precipitation and if i followed that, i'd only be left with 2 components. One more thing-there is a note on file (im doing this on behalf of someone not here currently that they used 50mL of supernatant concentrated down to 100uL to detect the protein on a western blot)..How do I get 50mL of supernant? Please help. I'm so confused



Ohh, was the 3rd component the secreted you think its to see if the protein has been secreted. In this case, would the TCA precipitation come from the supernatant in step number 3 (after pelleting my OD 0.6 culture?)