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Hi everybody,
Does someone of you know how to inactivate specifically PNGF?
I use PNGF to deglycosylate my protein of interest in native conditions. After this treatment I want to use my protein (deglycosylated) in some activity assays, but I want to inactive PNGF before do it, because it is quite possible that this enzyme interferes in my assays. Do you know any method to do it?
Thanks
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Posted By Pippuri
on 4/27/2011 6:59 AM
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Hi calandino77,
Boiling (100deg) for 10 to 15 min will do.
Good luck.
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Hi Pipurri,
Thanks again for your help. One question, wouldn´t boiling also denaturate my protein? becuase it is a complement system protein.
Greetings
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Posted By Pippuri
on 4/27/2011 8:22 AM
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Oops, sorry. I wasn't thinking about the assay part. Obviously heat-inactivation won't work.
As for now, the only way that I could think of is to use some sort of chromatography to separate them....
Now, we need to know a few more info- For example, What is the Mw of your protein of interest? Does your protein of interest carries any affinity tag?
Last edited Apr 27, 2011, 10:23 AM by Pippuri
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Posted By Suola
on 4/27/2011 17:33 PM
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calandino77,
You can try to chemically deglycosylate your protein with TFMS (which removes all N- and O-linked complex glycans), but this requires dissolving the protein afterwards in high molarity urea or Gnd-HCl, and then slowly renaturing the protein by for example dialyzing the denaturant away... This way you don't have to worry PNGF contamination. Both TMSF hydrolysis and protein denaturation-renaturation are used commonly in biochemical research.
Here's a protocol for the TFMS deglycosylation from Pam Stanley's Lab
Cheers,
Certainty of death, small chance of success... What are we waiting for? - Gimli
Last edited Apr 27, 2011, 20:26 PM by Suola
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Hi pipurri and suola,
Thanks for your help. I still don´t know which way to take but now at least I have some options. Thanks again for your time and help.
Greetings
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use .1% TFA or .1% Formic acid
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For clarification TFA and Formic acid with quench the PNGase F reaction and if you are doing mass spec that are excellent ion pairing agents. If you want to go further you can remove inactivated PNGase F by filtration over a microcon YM-30 centrifugal filter (Millipore, Billerica, MA).
You can also stop the reaction by adding 1 M HCl (~5 ul per 100 ul reaction volume)
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