FragIT kit

FragIT™ kit

FragIT kit is used for the preparation and purification of F(ab')2 and Fc/2 fragments from IgG. The kit consists of spin columns of FragIT (Immobilized FabRICATOR) for antibody digestion and spin columns of CaptureSelect™* Fc resin for affinity purification of the Fc fragments. 

FabRICATOR (IdeS) is a cysteine protease that digests antibodies at a specific site below the hinge, generating a homogenous pool of F(ab')2 and Fc/2 fragments after 30 min incubation.

Detailed Information

FabRICATOR digests IgG at a specific site below the hinge region, generating a homogenous pool of F(ab')2 and Fc/2 fragments. The IgG is digested within 30 minutes, and there is no risk of overdigestion if the incubation time is prolonged. Since FabRICATOR digests IgG under physiological reaction conditions, the immunoreactivity is preserved.

FragIT is a resin with FabRICATOR enzyme covalently coupled to agarose beads for generation of F(ab’)2 and Fc/2 fragments without contaminating the final preparation with enzyme. The workflow is simple - IgG is incubated with the FragIT resin and fragments are easily collected by a centrifugation step. The FragIT kit consists of FragIT resin for antibody digestion and CaptureSelect™* columns for affinity binding of the Fc fragments (other CaptureSelect™* resin needed for rabbit IgG, please inquire).

The FragIT kit is available for digestion and purification of 0.5 mg up to 100 mg of antibody or Fc-fusion protein.

FragIT digests all subclasses of human, and some classes of monkey, rabbit and sheep IgG. It has limited activity on mouse IgG2a and IgG3 - for digestion of these antibody species, we recommend using FragIT Z .

Storage and Stability

The FragIT spin columns and CaptureSelect™* spin columns are supplied in 20% ethanol, shipped cold, and should be stored at +4-8°C upon arrival. Do not freeze the products!

Buffers

FragIT can be used with all commonly used buffers with pH ranging from 6.0 to 8.0. Optimization may be required. Note that the FabRICATOR enzyme is irreversibly inactivated below pH 5.0.

Unit Definition

One spin column contains sufficient FabRICATOR-coupled agarose beads to digest 0.5 mg (Microspin), 10 mg (Midispin), or 100 mg (Maxispin) IgG.

*Thermo Scientific™ CaptureSelect™ resin from Thermo Fisher Scientific Inc. and its subsidiaries. Thermo Fisher and CaptureSelect are trademarks of Thermo Fisher Scientific Inc. and its subsidiaries.

FragIT-kit

  • A2-FR2-005

    FragIT kit, Digest and Purify up to 0.5 mg

    A2-FR2-005400.00Add to cart
  • A2-FR2-025

    FragIT kit, Digest and Purify up to 5 x 0.5 mg

    A2-FR2-0251,070.00Add to cart
  • A2-FR2-050

    FragIT kit, Digest and Purify up to 10 x 0.5 mg

    A2-FR2-0502,040.00Add to cart
  • A2-FR2-100

    FragIT kit Midispin, Digest and Purify 1-10 mg

    A2-FR2-1001,340.00Add to cart
  • A2-FR2-1000

    FragIT kit Maxispin, Digest and Purify 10-100 mg

    A2-FR2-10004,010.00Add to cart

How to Order

For details on ordering please visit How to order. Click add to cart to buy online.

Quotations

Please do not hesitate to contact us if you need a quotation or have inquiries on price of bulk quantities.

Documents

FAQ and Support

Popular FAQ

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.

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.

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.

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.

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.

 

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