Frequently Asked Questions
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Can OmniGLYZOR be used for released glycan analysis?
No, both N- and O-glycans are trimmed by the exoglycosidases present in OmniGLYZOR and do not longer represent the structures found on the intact glycoprotein substrate.
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Does OmniGLYZOR remove all N-glycans?
OmniGLYZOR hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.
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Is it possible to reuse the OmniGLYZOR column?
No, it is not recommended. We can only guarantee optimal performance for one-time use.
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Which buffers are compatible with IgMBRAZOR?
The enzyme is active at pH 5.5 to 9.0, tolerates up to 300 mM NaCl, and does not require reducing conditions or co-factors for activity. PBS and TBS buffers are both compatible. Optimization may be required if buffers other than the recommended are used.
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Is it possible to use IgMBRAZOR with very low concentrations of IgM?
It is possible to use IgMBRAZOR with low concentrations of IgM. However, this will lead to a decreased reaction rate, so for IgM concentrations below 0.5 mg/mL it is recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of overdigestion and the incubation time can be prolonged to overnight if necessary.
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Can IgMBRAZOR and PNGase F be used in the same reaction?
Yes, IgMBRAZOR and PNGase F can be used in combination if the reaction is performed under non-denaturing conditions. However, for complete deglycosylation of IgM using PNGase F denaturing conditions may be required and it is then recommended to perform the IgMBRAZOR digestion prior the PNGase F deglycosylation.
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Can IgMBRAZOR and FabRICATOR be used in the same reaction?
Yes, IgMBRAZOR and FabRICATOR can be used in combination.
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Does IgMBRAZOR digest only human IgM?
IgM from some species of monkey may also be digested by IgMBRAZOR.
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Are there any known inhibitors of ImpaRATOR?
ImpaRATOR is a metalloprotease and thereby sensitive to chelating agents such as EDTA. Concentrations > 1 mM EDTA results in complete inhibition of the enzyme. In addition, the ImpaRATOR activity is inhibited by reducing agents and detergents.
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Can ImpaRATOR be used under denaturing conditions?
Insufficient digestion during non-denaturing conditions can be caused by O-glycosylation sites within the glycoprotein inaccessible to the enzyme. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, buffer-exchange, and then digestion with ImpaRATOR. If detergent is added, make sure to include a detergent removal step prior ImpaRATOR digestion.
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