Inactivation of PNGF

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calandino77
calandino77's picture
Inactivation of PNGF

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

Chin Fen Teo
Chin Fen Teo's picture
 Hi calandino77,

 Hi calandino77,

Boiling (100deg) for 10 to 15 min will do.

Good luck.

calandino77
calandino77's picture
Hi Pipurri,

Hi Pipurri,
Thanks again for your help. One question, wouldn´t boiling also denaturate my protein? becuase it is a complement system protein.
Greetings

Chin Fen Teo
Chin Fen Teo's picture
Oops, sorry. I wasn't

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?

Sami Tuomivaara
Sami Tuomivaara's picture
calandino77,

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,

calandino77
calandino77's picture
Hi pipurri and suola,

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

ncwildb88
ncwildb88's picture
 use .1% TFA or .1% Formic

 use .1% TFA or .1% Formic acid

ncwildb88
ncwildb88's picture
 For clarification TFA and

 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).

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text-autospace:none">You can also stop the reaction by adding 1 M HCl (~5 ul per 100 ul reaction volume)