I'm trying to analyse the secreted proteins from a culture of bacteria, at the moment my current protocol is :
1. Grow 50mL cultures to about OD600 of 0.5-1.0.
2. Spin the cells down at 4,500rpm.
3. Filter the 50ml culture supernatant with 0.2um syringe-driven filter to remove any unpelleted bacteria, debris etc.
4. I then add deoxycholic acid (DOC) to a final concentration of 0.2mg/ml to the filtered supernatant and incubate on ice for 30mins.
5. After, I add 100% TCA to give a final concentration of 10%. And incubate on ice in a refrigerated cold room overnight. At this point the samples are cloudy.
6. The day after I then transfer the 30mL of the culture supernatant-doc-tca mixture into sterilized 30ml beckman centrifuge tubes (the oak ridge high x g ones) and centrifuge at about 13,000 rpm in a Beckman high capacity centrifuge.
7. Remove the supernatant.
8. I centrifuge the remaining 20mL of culture supernatant-doc-tca mixture as above.
9. Remove the supernatant and add 2mL of -20 acetone. And incubate at -20 for 1 hour.
10. Vortex and transfer into a 2mL collection tube.
11. Centrifuge at 14,000 rpm in a micro-centrifuge.
12. Remove supernatant.
13. Air dry.
14. I re-suspend the pellet (which is normally tiny and white, or not there at all) in 50-150uL of 0.1M sodium phosphate buffer + 1% SDS.
- Here's where hit a problem, sometimes after the first centrifugation (step 5) I find a white precipitate collect at the bottom after centrifugation, but this that wont pellet properly. I've tried centrifuge at 20,000 rpm, but the still happens.
- Another problem i have is that it seems I'm losing alot of proteins when I transfer the 2mL acetone into a eppendoorf for the 2nd centrifuge step (step 9-11).
- Also sometime the pellet wont dissolve very well (step 14).
- I run the samples on a 12% SDS-PAGE gel, and the results are often mixed, sometimes the protein band is pretty good, other times its awful (just 2-3 bands, compared to multiple bands).
Can anyone help with these problems? Or suggest alternations to my above protocol?
I've thought about doing the Acetone precipitation and Chloroform/Methanol precipitation methods, but these require larger volumes of liquids as I have a large starting volume of culture supernatant, so gets awkward when centrifuging.