PNGase F Glycan Cleavage Kit

PNGase F Glycan Cleavage Kit - FAQs

View additional product information for PNGase F Glycan Cleavage Kit - FAQs (A39245)

5 product FAQs found

In the PNGase Glycan Cleavage Kit, can I use PNGase F under native or denaturing conditions?

Yes, PNGase F enzyme may be used in both native and denaturing conditions

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In the PNGase Glycan Cleavage Kit, how much PNGase F should I use to remove my carbohydrate under native or DTT denaturing conditions?

When the protein is not denatured with SDS, PNGase F has to work harder to reach the cleavage site of the carbohydrate (due to secondary and tertiary protein structures). In these instances, using more enzyme and/or extended incubation times may help. These conditions will need to be determined empirically for your specific protein.

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In the PNGase Glycan Cleavage Kit, is PNGase F compatible with downstream analysis such as HPLC and mass spectrometry?

PNGase F enzyme is stored in 50% glycerol. Therefore, HPLC and/or mass spectrometry is not compatible due to the fact that glycerol is not tolerated in instruments used for these applications.

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In the PNGase Glycan Cleavage Kit, what are the typical reaction conditions for PNGase F, Recombinant?

Typical reaction conditions are as follows:

Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
1. Combine 1-20 µg of glycoprotein, 1 µL of 10X denaturing buffer (5% SDS, 400 mM DTT) and H2O (if necessary) to make a 10 µL total reaction volume.
2. Denature glycoprotein by heating reaction at 100 degrees C for 10 min.
3. Make a total reaction volume of 20 µL by adding 2 µL PNGaseF 10X Buffer, 1-5 µL PNGaseF enzyme, 2 µL 10% NP-40 and H2O.
4. Incubate reaction at 37 degrees C for 1 hr.
Note: Reactions may be scaled-up linearly to accommodate larger reaction volumes.

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In the PNGase Glycan Cleavage Kit, what happens to the asparagine residue after PNGase F removes the sugar?

As PNGase F is a glycoamidase, the asparagine gets converted into aspartic acid.

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