I'm wondering if anyone has had great success with any particular type of buffer for extracting mitochondrial proteins from purified whole mitochondria. I'm relatively new to the protein game, and I'm often not getting a consistent clear mt profile when run out on a gel.
My target protein has a predicted high kDa. I read that Urea is good for the isolation of high molecular weight proteins. The original protocol called for boiling the sample for 2 minutes, but I've conversely read heating urea will lead to cyanates.
Current Buffer - 9M Urea, 1% SDS, 25mM Tris HCl pH 6.8, 1mM EDTA, 0.7 BME.
Mt pellets are resuspended in 0.2mL's, incubated for about 30 minutes at RT, centrifuged and mixed with loading dye 6-1 (also not sure if I need an SDS based loading buffer or if the dye is sufficient alone, I've read mixed things on that front too).
Side question - I'm working with an ascomycete and sometimes pigments follow through with the whole mt extraction. In the urea buffer, the samples retain different gradations of color when everything is solubilized. As such, I'm unable to quantify them with BCA. Is there a way to overcome this / a different assay I should be using?
Hi, I´m working with plants and we also purified mitochondria and performed MS analysis we made good experience with a urea based buffer, we also performed sequential extractions with different urea based buffers. Here you can find the composition:
doi: 10.1007/s00299-010-0935-4
Further, heating of protein sampples is absolutely not recommended if you want to run MS analysis because it will lead in artificial carbamoylation of aminogroups. this will mask proteins from detection. also if thiourea is contained in the buffer it will hydrolyse your proteins.
Additionally, we have good experience in combining ruea based protein extraction with methanol/chloroform preciptation as we have to get rid of the chlorophyl. this works very satisfying.
Anyway your buffer composition does not (at least not for me) appear unsuitable but maybe its worth to include methanol/chloroform precipitation additionally.
Please let me know if this is helpful or if you have further questions.
cheers
Sebastian
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