Question 1

Is there an enzyme for digestion of IgA2-only?

Maria Ekemohn · Accepted Answer

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.

Question 2

What enzyme should be selected for the digestion of IgA1?

Maria Ekemohn · Accepted Answer

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.

Question 3

Which buffers are compatible with IgASAP Sub1?

Maria Ekemohn · Accepted Answer

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.

Question 4

Can the IgASAP Sub1 digestion be performed at lower temperatures?

Maria Ekemohn · Accepted Answer

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

Question 5

Does IgASAP Sub1 digest human IgA1-only?

Maria Ekemohn · Accepted Answer

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.

Question 6

Does IgASAP Sub1 digest human IgA1-only?

Maria Ekemohn · Accepted Answer

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.

Question 7

Can I digest rat IgG2a with FabRICATOR Z?

Felicia Mejare · Accepted Answer

Unfortunately, FabRICATOR Z does not digest rat IgG2a. If generating Fab fragments is an option, we suggest using FabULOUS enzyme that digests rat IgG2a. FabULOUS does not have a single specific cleavage site, but a primary digestion site, so there can be more than one digestion site. The digestion site(s) for rat IgG2a using FabULOUS is not determined, but the generated fragments are Fab and Fc.

Question 8

Can I re-use FabRICATOR Z Immobilized columns?

Felicia Mejare · Accepted Answer

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Question 9

Can I digest all mouse IgG subclasses with FabRICATOR Z?

Felicia Mejare · Accepted Answer

No, FabRICATOR Z only digests mouse IgG2a (….CPAPNLLG / GPSVF.. ) and IgG3 (…CPAPNILG / GPSVF…) at one specific site below the hinge. If Fab fragments are an option, we recommend our FabULOUS enzyme that digest mouse IgG1 above the hinge. FabULOUS requires light reducing conditions for optimal activity. If strong reducing conditions are used, this may likely cause reduction of the interchain disulfides.

Question 10

Can I release Fc fragments from the CaptureSelect column after isolating the F(ab')2 fragments?

kk · Accepted Answer

It is possible to elute the Fc fragments from the CaptureSelect column using a low pH buffer, for example a 0.1M Glycine buffer pH=3.0. The pH must immediately after elution be adjusted to neutral pH by adding 0.1x elution volume of 1M Tris pH=8.0.

Question 11

Can I release Fc fragments from the CaptureSelect column after isolating the F(ab')2 fragments?

kk · Accepted Answer

It is possible to elute the Fc fragments from the CaptureSelect column using a low pH buffer, for example a 0.1M Glycine buffer pH=3.0. The pH must immediately after elution be adjusted to neutral pH by adding 0.1x elution volume of 1M Tris pH=8.0.

Question 12

Can I re-use the GlycINATOR Immobilized column?

Felicia Mejare · Accepted Answer

We can only guarantee optimal deglycosylation for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Question 13

Can I achieve Fc-specific deglycosylation on IgG with N-linked glycans in the Fab region?

Felicia Mejare · Accepted Answer

Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.

Question 14

Which should I choose, GlycINATOR or IgGZERO?

Felicia Mejare · Accepted Answer

Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 15

What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?

Felicia Mejare · Accepted Answer

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

Question 16

What is the difference between GlycINATOR and IgGZERO?

Felicia Mejare · Accepted Answer

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 17

What is the difference between GlycINATOR and IgGZERO?

Felicia Mejare · Accepted Answer

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 18

Can I use PBS 1X instead of the IgGZERO reaction buffer?

Felicia Mejare · Accepted Answer

Yes, the IgGZERO enzyme is compatible with most commonly used buffers with pH ranging from 6.0 to 8.0, but the reaction conditions might need to be optimized to ensure optimal deglycosylation.

Question 19

I read that cleavage buffer for IgGZERO is 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 Can I use PBS 1X in place of cleavage buffer?

kk · Accepted Answer

Yes, many buffers are compatible with IgGZERO. Please keep pH close to neutral.

Question 20

I did not get complete deglycosylation using IgGZERO Immobilized, how can I improve digestion?

kk · Accepted Answer

First you could increase incubation time and increase temperature to 37°C. One explanation for incomplete deglycosylation with IgGZERO Immobilized could be that the antibody has higher amounts of high mannose, hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. High mannose glycans are particularly resistant to digestion recommend you try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in mAbs.

Question 21

What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?

Felicia Mejare · Accepted Answer

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

Question 22

Which should I choose, GlycINATOR or IgGZERO?

Felicia Mejare · Accepted Answer

Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 23

Which should I choose, GlycINATOR or IgGZERO?

Felicia Mejare · Accepted Answer

Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 24

Is it possible to purchase PNGaseF Immobilized in bulk?

Hanna Toftevall · Accepted Answer

Yes! Contact us at info@genovis.com

Question 25

Is it possible the reuse the  PNGAse F Immobilized column?

Hanna Toftevall · Accepted Answer

No, it is not recommended. We can only guarantee optimal performance for one-time use.

Question 26

Can the deglycosylation on the column be performed under denaturing conditions?

Hanna Toftevall · Accepted Answer

The columns are recommended to be used for deglycosylation at native conditions only.

Question 27

Does PNGase F  Immobilized remove all N-linked glycans?

Hanna Toftevall · Accepted Answer

PNGase F Immobilized 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.

Question 28

Which buffers can be used with the PNGaseF Immobilized column?

Hanna Toftevall · Accepted Answer

The recommended buffer for best performance of the column is TBS buffer at pH 8.6. Other buffers with neutral pH containing 0.15 M NaCl can also be used but optimization may then be required.

Question 29

Which buffers can be used with the PNGaseF Immobilized column?

Hanna Toftevall · Accepted Answer

The recommended buffer for best performance of the column is TBS buffer at pH 8.6. Other buffers with neutral pH containing 0.15 M NaCl can also be used but optimization may then be required.

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