Frequently Asked Questions
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I did not get complete deglycosylation using IgGZERO Immobilized, how can I improve digestion?
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.
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Is cysteine really needed for activation of the cysteine protease if TCEP or DTT is added in the reaction?
Yes, cysteine is required for both activity and specificity of the GingisREX enzyme.
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Is the reaction ready in only 60 minutes?
The digestion time is affected by many factors like the enzyme to sample ratio, pH of the digestion reaction, and the sample characteristics. The reaction time needs to be optimized for each specific case.
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Can guanidinium hydrochloride be used as a denaturing agent instead of urea?
No, GingisREX is inhibited by GdHCl even in low concentrations. This chaotrope should be avoided.
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Can you recommend a protocol for purification of the Fab?
Genovis provides a GingisKHAN Fab kit containing a specific Capture Select CH1 resin for further purification of the Fab. See this page for more information.
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Can I use another reducing agent besides GingisKHAN reducing agent?
We recommend using the 10x reducing agent provided with GingisKHAN which has an optimized pH to provide full enzymatic activity without affecting the disulphide bridges between the light chain and Fd region of the heavy chain, keeping the Fab intact. If other reducing agent is used there is a risk for non-optimal reaction conditions. It is important to use freshly prepared Reducing Agent (max storage 6h) for guaranteed activity of GinigsKHAN.
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How can I stop the GingisKHAN reaction?
The enzyme is a cysteine protease that can be inhibited by alkylating agents such as iodoacetamide. There are also several other known inhibitors to this enzyme, like KYT-36.
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Is GingisKHAN an IgG specific protease?
No, GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at one specific site (…KSCDK/THTCPPCP…) above the hinge, generating Fab and Fc.
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Can I use protein A or MabSelectSure (GE) for removal of Fc-fragments?
Genovis has chosen CaptureSelect in the FabRICATOR Fab2 Kit for removal of Fc fragments. CaptureSelect has a very high specific affinity for the Fc. Both protein A and MabSelect Sure (GE) may have affinity for the Fab which may result in a lower recovery of the generated F(ab’)2 fragments and the degree of affinity is antibody dependent. We have many customer reports on this subject, and they usually come back to us for the CaptureSelect columns for the above reason.
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Can I release Fc fragments from the CaptureSelect column after isolating the F(ab’)2 fragments?
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.
Learn more about FabRICATOR®