Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

How do you deproteinise blood samples?


  • Please log in to reply
2 replies to this topic

#1 Esther_23

Esther_23

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 19 February 2009 - 10:47 AM

Hi,

I have a very basic question that I have been unable to find an answer to so far - I will be collecting a series of very small blood samples (1.5ml) that I need to deproteinise for ammonia analyis. All the methodology I have read says to precipitate the proteins in ice-cold PCA or TCA (no problem) and centrifuge (also no problem!) then neutralise the supernate by drop-wise addition of KOH. This is where I come undone! How do you know when you have added sufficient KOH - a colour indicator would surely interfere with the subsequent ammonia assay and a pH probe seems too bulky for such tiny little samples. Since I am unable to find any information about this, I can only conclude that it is something so obvious that it doesn't need to be written and I must be really stupid!

Any advice that you can give me would be so very much appreciated!

Thanks, E

#2 NemomeN

NemomeN

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 34 posts
0
Neutral

Posted 19 February 2009 - 11:40 AM

Hi,

I have a very basic question that I have been unable to find an answer to so far - I will be collecting a series of very small blood samples (1.5ml) that I need to deproteinise for ammonia analyis. All the methodology I have read says to precipitate the proteins in ice-cold PCA or TCA (no problem) and centrifuge (also no problem!) then neutralise the supernate by drop-wise addition of KOH. This is where I come undone! How do you know when you have added sufficient KOH - a colour indicator would surely interfere with the subsequent ammonia assay and a pH probe seems too bulky for such tiny little samples. Since I am unable to find any information about this, I can only conclude that it is something so obvious that it doesn't need to be written and I must be really stupid!

Any advice that you can give me would be so very much appreciated!

Thanks, E


Using an extra tube, scale up your procedure (you can use culture media instead of blood) so that you can use either pH paper or meter when you do the neutralization part. Once you find out how much KOH to add (for the large scale procedure) you can then calculate back down to how much you would need for your smaller samples.

Good Luck

#3 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,805 posts
133
Excellent

Posted 20 February 2009 - 06:50 AM

if you have a large enough volume then you can take a droplet from the tube and apply it to the pH paper (instead of dipping the paper).

alternatively, you can use a microelectrode (we got one from microelectrodes, inc). you can use them in volumes less than 10ul.
talent does what it can
genius does what it must
i do what i get paid to do




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.