I am working on isolation of the total proteome of soils with a high organic content. There are very few labs which do this successfully. I was wanting to know the most preferred techniques for the isolation of the proteins (total proteome) and the bioinformatic processes used to translate protein identifications to meaningful results. I would also like to visit a lab that specializes in soil proteome isolation and learn some of their techniques and bioinformatic processes. All recommendations gratefully appreciated.
the preference is relative, if one thing works in your lab, you have to stick with it. also protein isolation depends on the question you want to address. cytoplasmic/soluble proteins are easier to deal with. membrane proteins will/are slightly difficult to isolate. How you are going to address the microbial fauna of the soil.
How much experience you have in this field. If starting from scratch I would weigh in a gram of soil. resuspend it in deionized water at 4 deg C for 2-24 hours . let the big particles settle. then start filtering the material using seives or alternatively you can use centrifugation to start pelleting the material. I would start at 500g and end at 50000g, the number of fractions will be your choice. more fractions more work. after collecting the pellets and supernatents and the final supernatent, analyze them using the SDS-PAGE.
Soil has lots of humic acids and other components. Dissolve in deionised water with mixting on magnetic stirrer for 1-2hrs and then centrifuge it at about 3000rpm. Take supernantant and then you centrifuge at high speed or ultracentifuge. Precipitate the proteins from supernant by acetone precipitation. You may add some lysis buffer to pellet, mix for 30 min (no foaming), centifuge, collect supernant. Determine protein contnet in both fractions and then analyse by SDS-PAGE.
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