Genovis » FragIT kit

Q: Can I re-use FragIT columns?

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.

Q: 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.

Q: Can I use FragIT kit for generation and purification of fragments from Rabbit IgG?

FragIT digests rabbit IgG but to capture the Fc fragments in the affinity step you need another resin than the Capture Select Fc normally included in the kit. Please contact the Genovis team if you are interested in processing rabbit IgG and we will provide columns with resin compatible with rabbit Fc in the FragIT kit.

Q: Can I use protein A or MabSelectSure (GE) for removal of Fc-fragments?

Genovis has chosen CaptureSelect in the FragIT 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.

Q: Does FabRICATOR introduce modifications?

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (...CPAPELLG / GPSVF...) and the C-terminus will regain the carboxyl group - that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H). In summary, R1-CO-NH-R2 + H2O --> R1-COOH + NH2-R2.

Description

FragIT kit contains immobilized FabRICATOR (IdeS) enzyme on agarose beads in spin columns and CaptureSelect^TM for affinity capture of Fc fragments. FragIT digests antibodies or Fc-fusion proteins at one single amino acid position below the hinge region generating Fc and F(ab’)2 fragments. CaptureSelect^TM spin columns capture Fc fragments for purification of F(ab’)2 and Fc fragments (other CaptureSelect^TM resin needed for rabbit IgG, please inquire). FragIT kit provides digestion and purification of 0.5 – 100 mg of antibody or Fc-fusion protein without residual enzyme in the final preparation.

Detailed Information

Application	Antibody Characterization, Antibody Fragmentation, Purify F(ab')2 and Fc Fragments
Product Group	FabRICATOR
Source/Origin	Cloned from S. pyogenes, Produced in E. coli (His-tagged), 37.7 kDa
Product Composition	Immobilized on agarose beads (20% EtOH), CaptureSelect Fc columns included
Digestion Site	Below Hinge
Specificity	CPAPELLG / GPSVF (below the hinge)
Substrates	Human IgG1, Human IgG2, Human IgG4, Monkey IgG, Rat IgG, Sheep IgG
Reaction Conditions	30 minute reaction at 37°C according to protocol specifications
Digestion Buffers	PBS (pH 7.4)
Buffer Compatibility	Other commonly used buffers at physiological pH (6.0 – 8.0) and ionic strength can also be used. Optimization may then be required.
Inhibitors	Iodoacetate 20 mM, Iodoacetamide 1 mM, pH < 5.1
Unit Definition	0.1 ml settled resin digests ≥ 95% of 0.5 mg Human IgG1

FAQs

Can I re-use FragIT columns?

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.

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.

Can I use FragIT kit for generation and purification of fragments from Rabbit IgG?

FragIT digests rabbit IgG but to capture the Fc fragments in the affinity step you need another resin than the Capture Select Fc normally included in the kit. Please contact the Genovis team if you are interested in processing rabbit IgG and we will provide columns with resin compatible with rabbit Fc in the FragIT kit.

Can I use protein A or MabSelectSure (GE) for removal of Fc-fragments?

Genovis has chosen CaptureSelect in the FragIT 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.

Does FabRICATOR introduce modifications?

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.

Where does FabRICATOR cleave the IgG molecule?

FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: HTCPPCPAPELLG / GPSVF
Human IgG2: VECPPCPAPP_VA / GPSVF
Human IgG3: PPCPRCPAPELLG / GPSVF
Human IgG4: PHAHHAQAPEFLG / GPSVF

FragIT kit, Digest and Purify up to 0.5 mg quantity	FragIT kit, Digest and Purify up to 0.5 mg (A2-FR2-005)	€400.00
FragIT kit, Digest and Purify up to 5 x 0.5 mg quantity	FragIT kit, Digest and Purify up to 5 x 0.5 mg (A2-FR2-025)	€1,070.00
FragIT kit Midispin, Digest and Purify 1-10 mg quantity	FragIT kit Midispin, Digest and Purify 1-10 mg (A2-FR2-100)	€1,340.00
FragIT kit Maxispin, Digest and Purify 10-100 mg quantity	FragIT kit Maxispin, Digest and Purify 10-100 mg (A2-FR2-1000)	€4,010.00

FragIT kit

Description

Detailed Information

FAQs

Can I re-use FragIT columns?

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

Can I use FragIT kit for generation and purification of fragments from Rabbit IgG?

Can I use protein A or MabSelectSure (GE) for removal of Fc-fragments?

Does FabRICATOR introduce modifications?

Where does FabRICATOR cleave the IgG molecule?

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