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GST ligand capture

  • zm00040
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1 year 6 months ago #1 by zm00040
GST ligand capture was created by zm00040
Hi there,
I'm trying to capture a GST-tagged ligand with the GST capture kit. The immobilisation of the Anti-GST Ab seemed to work fine giving ~11,000RU. However when I try to capture the GST-ligand and recombinant GST as the negative control, i only generated RU of ~100 (I then tried again for longer contact time to 420s and doubling the protein conc. to 20ug/mL, but still RU ~100). Then when I run small molecules over the chip, I see no interactions with the ligand as I should expect. Any advice/things I should try?
Many thanks
FC2 immob
FC1 immob
Recombinant GST capture
GST-tagged protein capture
Binding sensorgram
  • reeve
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1 year 5 months ago #2 by reeve
Replied by reeve on topic GST ligand capture
Hi,
I am not familiar with the instrument you use, and I could not see the picture about FC1 and FC2 clearly, but looks like you immobilized GST-Ab to both FC1 and FC2, so there was no reference chamber? If that was the case, the relative response can only be that small. Please immobilize GST-Ab in only one chamber and make the other the reference.
Best,
  • kenley
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3 months 2 weeks ago #3 by kenley
Replied by kenley on topic GST ligand capture
Hi there,
I was wondering if you found the reason for the very low capture level? I am getting the same results as you saw previously so it would be great if you have any theories to solve this. Thanks
  • zm00040
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3 months 1 week ago #4 by zm00040
Replied by zm00040 on topic GST ligand capture
Hi Kenley,
I still wasn't sure what was causing the low level of capture. It may well have been as Reeve said because I immobilised GST-Ab to both FCs but I wanted to do this for better CTL (ctl FC then had GST bound to it). The solution was I performed the GST capture 3 times instead of just once to sufficiently bind the protein and this seemed to then give better responses. Sorry I couldn't be of more help.
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