Membrane Protein Extraction and Analysis Questions - (Dec/18/2007 )
I have a thermophilic iron and sulfur oxidizing archaea with a genome that is almost complete. Genetic analysis has yielded many novel electron transport chain genes with possible roles in s and iron oxidation. I have had great success with RNA expression and DNA work.
I am just now going to enter the proteomics world. How difficult will it be to extract membrane proteins from this organism (grows optimally at 70 C on pyrite up to 10 e9 cells/ml)?
Once the proteins are extracted, I envision fractioning them (with HPLC??) and finding fractions that oxidize iron. I would then analyze these fractions for spectra and maybe maldi-tof.
My questions are: Am I on the right track? Is this a doable scenario for someone who has don no protein work but has access to others labs with the equipment. How difficult is it to do these steps? What equipment do I need to find? What journal articles and method papers, websites could one steer me to.
I would also like to do a 2-d gel with cells grown on sulfur vs. cultures on pyrite to see differences. How hard is this process and is it easy to analyze these proteins?
I want as much info as possible before I go to the biochemists. I do not want to appear unprofessional and unprepared as I am gearing up on my last year of a PhD program.
Mark at Montana State University
First,you should extraction the total protein and then HPLC (SCX and RPLC)