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1、 m 1 Dilute anti GST antibody to 30 g ml in immobilization buffer for example 3 l anti GST 77 l immobilization buffer for one 7 min immobilization at a flow rate of 5 l min k W Use the solution within 12 h 2 Immobilize the diluted anti GST antibody using the Amine Coupling Kit Recommended conditions
2、 are flow flow rate 5 10 l min 6 7 min activation with EDC NHS 7 min immobilization of anti GST antibody 7 min deactivation with ethanolamine A typical immobilization level using these conditions at 25 C is 10000 RU The immobilized amount is not critical for capturing GST fusion proteins To adjust t
3、he immobilization level vary the contact time for activation of the surface Activity Control Recombinant GST is provided for checking the activity of the immobilized anti GST antibody m 1 Dilute recombinant GST to 5 g ml in HBS EP buffer 3 Inject the appropriate volume of diluted recombinant GST ove
4、r the anti GST surface at 20 l min flow rate using HBS EP buffer as running buffer The response typically 1000 1500 RU with 120 l on 10 000 RU immobilized anti GST antibody provides a measure of the surface capacity The dissociation rate at 180 s after the end of the injection should not exceed 3 RU
5、 min see Figure 1 4 Regenerate the surface with Regeneration solution for 2 min at 20 l min Figure 1 Sensorgram obtained from injection of recombinant GST over an anti GST surface Recommended Analysis Procedure Perform a positive control e g activity control using recombinant GST as the first cycle
6、on a newly immobilized surface The first injection will often give a higher response than the following cycles A GST fusion protein concentration of 10 g ml or less is recommended After injecting GST fusion protein wait until the response is stable before injecting binding partner A wait time of 180
7、 s or less is in most cases sufficient Regeneration Procedure Regeneration removes the GST fusion protein and any binding partner leaving anti GST antibody available on the surface For 5 out of 7 fusion proteins tested regeneration was satisfactory with a 2 minute injection of regeneration solution
8、at a flow rate f 20 l min although a baseline increase of 15 20 RU cycle could not be avoided in some cases For proteins where these conditions do not give adequate regeneration the following conditions are suggested 1 min with regeneration solution followed by 1 min with 0 05 SDS at a flow rate f 2
9、0 l min or Addition of up to 30 ethylene glycol to the Regeneration solution 20000 20500 21000 21500 020040060080010001200 Time s Response RU dissociation rate 3 RU min End of injection Page 3 4 Page 2 4 fkpqor qflkp clo rpb fkpqor qflkp clo rpb GST Capture Kit Product Description Order code BR 1002
10、 23 Contents Goat anti GST antibody immunosorbent purified and sterile filtered 0 8 mg ml in 0 15 M NaCl 75 l Recombinant GST p sterile filtered 0 2 mg ml in HBS EP buffer 10 mM HEPES pH 7 4 0 15 M NaCl 3 mM EDTA 0 005 Surfactant P20 100 l Immobilization buffer 10 mM sodium acetate pH 5 0 5 ml Regen
11、eration solution 10 m M glycine HCl pH 2 2 70 ml Storage 4 8 C For use only GST Capture Kit is designed to provide site directed capture of GST fusion proteins on immobilized anti GST antibody allowing further studies of interactions between the fusion protein and binding partners Binding of fusion
12、proteins to the anti GST antibody is stable Typical dissociation rate 3 RU min at a binding level of 1000 RU Recommended Immobilization Procedure o Amine Coupling Kit available from Biacore HBS EP running buffer available from Biacore Sensor Chip CM5 available from Biacore 2 For details on how to co
13、ntact your local Biacore representatives please visit us at Head Office Biacore AB Rapsgatan 7 SE 754 50 Uppsala Sweden Phone 46 18 675700 Fax 46 18 150110 Biacore is a registered trademark of Biacore AB Copyright Biacore AB GST Capture Kit Instructions for Use February 16 2005 Page 1 4 fkpqor qflkp clo rpb fkpqor qflkp clo rpb Page 4 4 22 0522 19 v3
