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Very Low Capture Levels
- Alluc.inazione
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5 years 3 weeks ago #1
by Alluc.inazione
Very Low Capture Levels was created by Alluc.inazione
Hi everyone,
I'm pretty new to SPR and I'm having quite some issue with my protein (ULK1) binding to the chip.
I'm trying to isolate it from mammalian cells and then perform SPR on a CM3 chip. I have two different tags on my protein of interest: (2X)His-FLAG-ULK1 and GST-(2X)His-ULK1.
I've tried to isolate it with GST IP, Ni-NTA IP and now I'm trying with FLAG. everytime I've loaded my purified protein on the machine, the capture levels were really low (100-400 RU max vs 5000-6000 RU we were expecting). of course after elution I've performed a buffer exchange before starting the experiment.
I also tried to use the whole cell lysate but as yo can imagine I got a lot of aspecific binding to the chip.
Do you have any idea on how I could improve my experiment?
Thank you
Alice
I'm pretty new to SPR and I'm having quite some issue with my protein (ULK1) binding to the chip.
I'm trying to isolate it from mammalian cells and then perform SPR on a CM3 chip. I have two different tags on my protein of interest: (2X)His-FLAG-ULK1 and GST-(2X)His-ULK1.
I've tried to isolate it with GST IP, Ni-NTA IP and now I'm trying with FLAG. everytime I've loaded my purified protein on the machine, the capture levels were really low (100-400 RU max vs 5000-6000 RU we were expecting). of course after elution I've performed a buffer exchange before starting the experiment.
I also tried to use the whole cell lysate but as yo can imagine I got a lot of aspecific binding to the chip.
Do you have any idea on how I could improve my experiment?
Thank you
Alice
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- Arnoud
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5 years 3 weeks ago - 5 years 3 weeks ago #2
by Arnoud
Replied by Arnoud on topic Very Low Capture Levels
Hi Alice.
You have to bear in mind that a CM3 has a shorter dextran matrix length than a CM5. Although they say that the carboxylation is the same as on a CM5 the CM3 has less spots to bind your protein and thus a lower capacity.
kind regards
Arnoud
P.S. lets continue in the other question
You have to bear in mind that a CM3 has a shorter dextran matrix length than a CM5. Although they say that the carboxylation is the same as on a CM5 the CM3 has less spots to bind your protein and thus a lower capacity.
kind regards
Arnoud
P.S. lets continue in the other question
Last edit: 5 years 3 weeks ago by Arnoud.
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