Is it TRIZol a good method to extract proteins? If so, which would be the best method to quantify proteins after TRIZol extraction? I'm planning to use the TRIzol reagent from ambion Life Technology (#15596-026), and BCA to quantify protein.
http://products.invitrogen.com/ivgn/product/15596026
http://www.piercenet.com/browse.cfm?fldID=02020101
If you only need the protein fraction (no RNA, no DNA) I would not use TRIzol for protein extraction. Depending on the proteins of interest (cytoplasmic, nuclear, membranous) and the analyses you plan to do on the protein extracts I would rather prepare DignamC, RIPA or Laemmli-Urea extracts. For most applications we prepare Laemmli-Urea extracts as it is fast and you retain proteins of all cell compartments. If we want to measure enzyme activity we prepare either DignamC or TNT extracts.
If you only need the protein fraction (no RNA, no DNA) I would not use TRIzol for protein extraction. Depending on the proteins of interest (cytoplasmic, nuclear, membranous) and the analyses you plan to do on the protein extracts I would rather prepare DignamC, RIPA or Laemmli-Urea extracts. For most applications we prepare Laemmli-Urea extracts as it is fast and you retain proteins of all cell compartments. If we want to measure enzyme activity we prepare either DignamC or TNT extracts.
I usually use the RIPA buffer and it works fine for me. The problem is that now I have to get both mRNA and protein for qRT-PCR and Western from the same sample (otherwhise I would have to increase my sample). Proteins I want to detect are nuclear and mitochondrial. Would u mind of sending me the refference of ur Laemmli-urea protocol to take a look, I don't know it. Thanks for ur reply!
I am also interested in the Laemmli-urea protocol. I currenlty use RIPA which does give a satisfactory protein yeild but from further research I have found that my protein target is also present in vesicle compartments (as well as in the plasma membrane). Thank you.
When we use Laemmli-Urea buffer for cell culture experiments we lyze 10^6 cells (fresh pellet or frozen) in 100µl of the buffer. As DNA is released you should cut the tip. Boil samples for 5min at 100°C. Load samples onto SDS gel or store at 4°C. Even after having stored the samples for more than one year at 4°C, I could still detect very instable proteins. Once I tried to seperate the samples on a Bis-Tris Gradient Gel (4-12%) which did not properly work (smear). The recipe is the following:
235 mM Tris (pH 6.8), 18.75% glycerol, 5.6% SDS, 6M urea, 0.002% bromphenol blue. Store at -20°C. Add 5% of 2-mercaptoethanol before use.
To extract proteins from tissue, I prepared the buffer without bromphenol blue and freshly added 1mM DTT (not 2-ME). I measured protein concentration with the BCA kit (microplate procedure, instead of 25µl of sample we usually use 2µl). Store these samples at -20°C or below.
Good luck for your experiments!
Hello pals!! Im interested in the tech too, I'll do it in this week... Im trying to purify a recombinant protein from E.Coli BL21 DE3 Plyss... Since I'd tried a kit from Qiagen where I'd have a bad result I want to test other technique... I was thinking in taking one pellet from a 25ml culture of BL21 and puting 1ml of Trizol, expecting in taking the DNA phase and making an agarose gel 1% to assure the gene expression and taking the organic phase and making a Western Blot or maybe only with Coomassie Blue or maybe with Biebrich Scarlet.... What do you guys think? Please help me in thoughts, I will appreciate it very much.
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