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). kW Use the solution within 12 h. 2. Immobilize the diluted anti-GST antibody using the Amine Coupling Kit. Recommended c
2、onditions 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 25C is 10000 RU. The immobilized amount is not critical for capturing GST fusion p
3、roteins. To adjust the 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
4、diluted recombinant GST over 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 i
5、njection should not exceed 3 RU/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 u
6、sing recombinant GST) as the first cycle 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
7、 injecting binding partner. A wait time of 180 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 w
8、ith a 2-minute injection of regeneration solution 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 solut
9、ion followed by 1 min with 0.05% SDS at a flow rate f 20 l/min, or Addition of up to 30% ethylene glycol to the Regeneration solution. 20000 20500 21000 21500 0 200 400 600 800 1000 1200 Time (s) Response (RU) dissociation rate 3 RU/min End of injection Page 3/4 Page 2/4 fkpqorqflkp=clo=rpb= fkpqorq
10、flkp=clo=rpb= GST Capture Kit Product Description Order code: BR-1002-23 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
11、% Surfactant P20), 100 l Immobilization buffer (10 mM sodium acetate, pH 5.0), 5 ml Regeneration solution (10 m M glycine-HCl pH 2.2), 70 ml Storage: + 4-8C For = use only. GST Capture Kit is designed to provide site-directed capture of GST fusion proteins on immobilized anti-GST antibody, allowing
12、further studies of interactions between the fusion protein and binding partners. Binding of fusion 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
13、 running buffer (available from Biacore) Sensor Chip CM5 (available from Biacore) = = 2 For details on how to contact 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 fkpqorqflkp=clo=rpb= fkpqorqflkp=clo=rpb= Page 4/4 22-0522-19 v3
