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Immobilized IdeS protease

Cat No.
NATE-1779
Description
NATE-1779 is a resin with IdeS protease covalently coupled to agarose beads for subunit fragmentation of IgG to generate pure F(ab’)2 and Fc fragments without contaminating the final preparation with enzyme. IgG is simply incubated with the NATE-1779 resin and fragments are then easily collected by a centrifugation step.
NATE-1779 cleaves IgG at one specific site below the hinge region and thereby eliminating the risk of over digestion if the incubation time is increased. NATE-1779 can be used with all commonly used buffers with pH ranging from 6. 0 to 8. 0.Optimization may be required.
NATE-1779 cleaves all subclasses of human, monkey, rabbit and sheep IgG but only subclass IgG2a and IgG3 of mouse IgG. Fragmention of Mouse IgG2a with NATE-1779 requires significantly longer incubation time as compared to with human IgG.
Abbr
IdeS protease, Immobilized (Streptocoocus pyogenes)
Source
E. coli
Species
Streptocoocus pyogenes
Product Overview
Additional Materials Required
• Cleavage buffer: 10mM sodium phosphate, 150mM NaCl, pH 7. 4 or similar with pH ranging from 6. 0-8. 0.
• Collection tubes: Micro centrifuge tubes (1. 5-2 ml).
Method
• Lids and bottom caps are used during the incubation.
• Before centrifugation remove the bottom cap and slightly open the lid ~90° counter clockwise.
Fragmentation
1. Make sure your antibody is in cleavage buffer (See Additional Material Required above).
2. Break off the bottom seal of the column and slightly open the lid ~90° counter clockwise.
3. Place the column in a 1. 5-2 ml collection tube.
4. Centrifuge the column at 200×g for 1min to remove storage solution.
5. Equilibrate the column by adding 300 µl cleavage buffer.
6. Centrifuge the column at 200×g for 1min.
7. Repeat steps 5 and 6 two times.
8. Put on the bottom cap on the column.
9. Immediately add the IgG to be cleaved in a volume of 100 µl at a maximal concentration of 5 mg/ml in cleavage buffer.
10.Seal the column with the top lid.
11. Take care to fully suspend the media manually and make sure it is flowing in the column.
12. Incubate the column by end-over-end mixing for 15min in room temperature. The incubation time can be increased without over digestion of the IgG. *For digestion of mouse IgG2a the incubation time needs to be significantly
increased, see note below.
13. Remove the top lid and the bottom cap.
14. Place the column in a 1. 5-2 ml collection tube.
15. Centrifuge the column at 1000×g for 1min to elute the sample.
For maximum recovery of your sample:
16. Place the column in a 1. 5-2 ml collection tube.
17. Add 100 µl cleavage buffer.
18. Centrifuge the column at 1000×g for 1min to elute the sample.
19. Repeat steps 16-18 one more time.
20.Pool all the elution fractions.
*Digestion of Mouse IgG2a.
For optimal fragmentation of mouse IgG2a the temperature can preferably be increased to 37°C and the incubation time needs to be optimized and can be increased to 6 – 48 hours.
Form
Each column include sufficient material to digest 0.5 mg IgG. It is supplied in 20% EtOH with no preservatives added.
Storage
It is shipped on ice and should be stored at +4-8°C upon arrival. Do not freeze the product!
Synonyms
IdeS protease; FabRICATOR; immobilized IdeS protease; immobilized FabRICATOR; FragIT
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