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Pierce IgG Fragmentation Kits FAQ


Answers to frequently asked questions (FAQs) about Pierce IgG Fragmentation Kits


 

In 2008, all three IgG Fragmentation Kits were redesigned and launched with new product numbers. How do the new kits differ from the previous ones?
The previous kits provided a single protocol for each immobilized enzyme, requiring researchers using different IgG volumes, concentrations or species to fully optimize the protocol themselves. The new kits are available in two formats (standard and micro) and optimized for fragmenting three species of IgG. The spin columns in the new kits provide more flexible protocols that are faster and easier and result in better yields. The kit format differences are summarized in the following table.

Comparison of the IgG fragmentation kit formats.

 

Previous Kits

New Kits

IgG amount

Single size: 10 mg
(1 mg for the IgG1 kit)

Micro kits: 25-250 mg (0.2-2 mg/ml)
Standard kits: 0.25-4 mg (0.5-8 mg/ml)

Protein A format

Gravity-flow column

Spin column

Desalting columns

None included

Desalting spin columns included

Digestion method

Glass test tubes, serum separators, mixed with a shaker

Spin columns, mixed with an end-over-end mixer or tabletop rocker

What species of IgG can be fragmented with these kits?
The protocols are optimized for rabbit, human and mouse IgG. Fragmentation of other IgG species may require optimization. To purify Fab or F(ab´)2 fragments from the digest, the IgG species must be able to bind to Protein A. For mouse IgG1, use a ficin-based kit (#44980, 44680).

How many kits are available and which enzymes are included?
There are six Thermo Scientific Pierce IgG fragmentation kits.

  • Fab Preparation Kit (Papain), #44985
  • Fab Micro Preparation Kit (Papain), #44685
  • F(ab´)2 Preparation Kit (Pepsin), #44988
  • F(ab´)2 Micro Preparation Kit, (Pepsin), #44688
  • Mouse IgG1 Fab and F(ab´)2 Preparation Kit (Ficin), #44980
  • Mouse IgG1 Fab and F(ab´)2 Micro Preparation Kit (Ficin), #44680

Can I purchase the immobilized enzymes (proteases) separately?
Yes. The three immobilized enzymes are available in 5 ml package sizes as follows:

  • Immobilized Ficin, #44881
  • Immobilized Papain, #20341
  • Immobilized Pepsin, #20343

When digesting an IgG that does not bind to Protein A, how do I purify the Fab and F(ab´)2 fragments?
If the IgG is a species or subclass that does not bind to Protein A, researchers can immobilize an antibody specific for the desired fragment. For example, to purify digested rat IgG2a, use the Aminolink Plus Immobilization Kit (#44894) to immobilize goat anti-rat Fc antibody. Our Tech Tip #34 and other web resources list IgG binding properties of Protein A. (Because Protein G and Protein A/G bind Fab fragments, they are not effective for this application.)

How long does IgG digestion take using these kits?
The digestion time is a function of the IgG concentration and source. For rabbit, mouse and human IgG, optimized digestion times are given in the instructions for each kit. Digestion times listed in the product instructions result in 80-90% digestion, although efficiency varies depending on IgG preparation and source.

Can I reuse the immobilized digestion enzymes?
No. For best results, use the immobilized enzyme once and discard after use.

 

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Instructions | MSDS | CofA
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