2 replies to this topic

[Laszlo]

Hi I'm new here.

My name is Laszlo Groh, I'm an undergrad in the Netherlands currently on an internship working in Paediatric Infectious Diseases research laboratory.

I'm developing a bioassay using HEK-Blue cells from InvivoGen to detect soluble MDP in nasopharyngeal aspirate samples (NPA) collected from childeren (not going into anymore details ).

MDP is composed of N-acetylmuramic acid linked by its lactic acid moiety to the N-terminus of an L-alanine D-isoglutamine dipeptide (according to N. Inohara et al.)

There appears to be some contamination in the NPA samples that we are testing, and so we'd like to pre-treat our samples with heat or irradiation (UV or otherwise) inactivation. Our main concern is that we would like to choose a methodology of inactivation that has the least effect on MDP's conformational state (which is essential for our assay) yet maximum protein denaturation/deactivation.

So my question is, how much can I beat up MDP before is becomes biologically inactive? Heat vs. UV/Irradiation

Cheers,
Laszlo Groh

[bob1]

I'm not aware that UV or even gamma irradiation has much affect on protein activity.  Heat certainly works, but you could also try increasing the salt concentration, denaturing agents such as DTT (which should affect proteins but not anything without cysteines), or sonication.  Perhaps treatment of your samples with a proteinase might work too (though proteinases might digest the MDP, I don't know enough to be able to tell)?

You might be best to get some MDP made and spike it into protein solutions to test the activity/conformation for yourself.

[Laszlo]

Hey,

We've managed to work this out in the lab. Alkaline phosphatases lose biological activity when heated at 65 Celsius for 30 minutes, and oddly enough MDP maintains biological reactivity when treated under the same condition. Although this is MDP in Mili-Q, not sure how a complex matrix like NPA may effect MDP at these temperature, but I doubt there will be an effect.

So now to heat all my samples!!