Is there an enzyme for digestion of IgA2-only?

There is no known enzyme that targets IgA2-only since the amino acids in the hinge regions of IgA2 is present also in IgA1. However, IgASAP Sub1 will digest IgA1-only.

What enzyme should be selected for the digestion of IgA1?

IgASAP Sub1 specifically targets IgA1 and exhibits a faster reaction time (1 hour) compared to IgASAP Sub1+2 (16-18 hours). The cleavage site of IgASAP Sub1+2 is located six amino acids above that of IgASAP Sub1, resulting in the absence of potentially O-glycosylated residues on the Fab fragments. This characteristic can be advantageous in certain cases.

Can the IgASAP Sub1 digestion be performed at lower temperatures?

The optimal activity is at 37°C, but the digestion can also be performed at room temperature using a prolonged incubation time.

Which buffers are compatible with IgASAP Sub1?

The enzyme is active at pH 6.5 to 8.0, 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.

Does IgASAP Sub1 digest human IgA1-only?

IgASAP Sub1 may digest IgA1 from other primate species with similar hinge sequences, but this has not been tested. IgASAP Sub1 does not digest IgA2.

Does ImpaRATOR cleave at all O-glycosylated sites?

ImpaRATOR has a broad activity towards different O-glycan structures; however, the enzyme has limited activity towards sites with two adjacent O-glycosylated Ser/Thr residues. For complete information about O-glycan sites in glycoprotein substrates containing several sites with two adjacent O-glycosylated Ser/Thr residues, OpeRATOR may be a better option.

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.

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.

Does IgMBRAZOR digest only human IgM?

IgM from some species of monkey may also be digested by IgMBRAZOR.

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