I would like to know if it is important to add protease inhibitor to plasma when using EDTA tube for a proteomic study? If so, what is the suggested brand? Thanks
Mona,
I spent years researching that exact topic. My opinion is that if you are doing biomarker discovery, you should do EVERYTHING you can to preserve the blood sample in as close to the in-vivo state as you can.
Protease inhibitors may be very important. The product I developed from those efforts was designed to give IMMEDIATE exposure to the inhibitors, with a cocktail optimized for blood. I'm not with that company anymore, and do not wish to 'advertise' -- but the product is called the P100 Blood collection tube, from Becton Dickinson. On the product webpage, there are links to lots of our data, showing details of the science itself.
Also be aware that blood has other ways to self-destruct. Spin the cells away as soon as possible --> the cells start to react to the tube environment, excreting proteins (including proteases), cytokines, and many small molecules that are definitely not native to the circulating plasma. Also be aware that the timeframe for some kinds of damage, from my experiments, can literally be just seconds after drawing the blood.
I also refer to the 2011 article that I co-authored (see my ResearchGate page), describing best practices for blood handling.
Good luck with your research. ---- Craig
Hi there,
If you want to avoid degradation of plasma proteins, of course add protease inhibitor cocktail. The only I use is Roche complete with or without EDTA in it.
Mona,
I spent years researching that exact topic. My opinion is that if you are doing biomarker discovery, you should do EVERYTHING you can to preserve the blood sample in as close to the in-vivo state as you can.
Protease inhibitors may be very important. The product I developed from those efforts was designed to give IMMEDIATE exposure to the inhibitors, with a cocktail optimized for blood. I'm not with that company anymore, and do not wish to 'advertise' -- but the product is called the P100 Blood collection tube, from Becton Dickinson. On the product webpage, there are links to lots of our data, showing details of the science itself.
Also be aware that blood has other ways to self-destruct. Spin the cells away as soon as possible --> the cells start to react to the tube environment, excreting proteins (including proteases), cytokines, and many small molecules that are definitely not native to the circulating plasma. Also be aware that the timeframe for some kinds of damage, from my experiments, can literally be just seconds after drawing the blood.
I also refer to the 2011 article that I co-authored (see my ResearchGate page), describing best practices for blood handling.
Good luck with your research. ---- Craig
Dear Dr. Craig A Gelfand,
Thank you so much for the reply. It was very informative. Yes, I am looking for biomarkers! And, thanks for introducing the "P100 Blood Collection Tube"
I would see If I could get this type in Iran! So, what you are saying is, with this kind of tube I would not require any protease inhibitor? and the tube itself provides essential inhibitors?
Thanks again for your time!
Yes, the tube has protease inhibitors included, so they dissolve immediately in the blood while it is being drawn. Since I'm not at BD anymore, I can't offer much help for how to get the tubes. Maybe the website has current contact info?
Perhaps more important in your case would be careful control and ability to guarantee repriducing every step in the blood collection and handling. I still told everyone that, even if they were using P100. My 2011 book chapter was written specifically to describe best practices. If you don't get P100, EDTA tubes would be my next choice -- which we describe in our JPR paper (2005, as I recall). Speed in handling from the vein to the -80 freezer is important also.
I would like to ask whether the following trypsin digestion step gets hindered by trypsin inhibition due to presence of the protease inhibitors.
Indeed lots of people were (and are) rightly concerned about that. In our hands, it was not enough to have much observable reduction in trypsin activity for typical peptide generation. That is so much extra trypsin that are added intentionally for peptide mapping experiments! As with all things, best to prove it doesn't alter your own methods though.
Dear Mona
If you used under liquid nitrogen for tissues or on ice for serums and prevent heat shock during sample preparation, the use of protease inhibitor is not mandatory.
The all proteins are denatured by using rehydration buffer in IEF.
I use no protease inhibitor cocktail and found very well 2-DE map. You can find it in Electrophoresis. 2014 Dec;35(23):3331-8.
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