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Negative Sensorgrams
- PJMCM
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3 months 2 weeks ago #1
by PJMCM
Negative Sensorgrams was created by PJMCM
Hello all,
I suspect I might have worked out the answer to my question in the process of putting this topic together, but I'd love to hear to some more thoughts on a problem I've been having. Please see the Slides 1 to 5 attached to help explain my process and problem.
In brief - I suspected that I was seeing non-specific binding of some tool compounds to the neutravidin surface I was using to capture my biotinylated protein of interest. To test this I ran parallel binding experiments in two different channels - one with only neutravidin immobilised and the second with neutravidin and then my protein captured onto this.
Crucially - in this second channel I ended up with more neutravidin in Fc1 than in Fc2.
For the compound I was most suspicious about ('Compound 3') I think I confirmed the above - clear binding response in the neutravidin-only channel and a negative binding response in the neutravidin/PoI channel (negative sensorgrams caused by the higher neutravidin in Fc1 being subtracted from a lower neutravidin response in Fc2).
For Compounds 1 and 2 I'm slightly more confused as I think I clearly see binding in the neutravidin only channel - but I don't see that same negative binding response in the neutravidin/PoI channel.
All sensorgrams/binding plots shown are buffer blank subtracted, where 1 % DMSO buffer (to match the 1 % DMSO content of all compound-containing injections) has been injected over the same surfaces.
Key questions:
1.) Why do we think I'm not seeing the negative binding responses for Compounds 1 and 2?
2.) Why am I not seeing the same binding responses/sensorgram profile between Channel 6's Fc2 and Channel 7's Fc1 (both should be neutravidin-only).
Many thanks for any thoughts and help!
I suspect I might have worked out the answer to my question in the process of putting this topic together, but I'd love to hear to some more thoughts on a problem I've been having. Please see the Slides 1 to 5 attached to help explain my process and problem.
In brief - I suspected that I was seeing non-specific binding of some tool compounds to the neutravidin surface I was using to capture my biotinylated protein of interest. To test this I ran parallel binding experiments in two different channels - one with only neutravidin immobilised and the second with neutravidin and then my protein captured onto this.
Crucially - in this second channel I ended up with more neutravidin in Fc1 than in Fc2.
For the compound I was most suspicious about ('Compound 3') I think I confirmed the above - clear binding response in the neutravidin-only channel and a negative binding response in the neutravidin/PoI channel (negative sensorgrams caused by the higher neutravidin in Fc1 being subtracted from a lower neutravidin response in Fc2).
For Compounds 1 and 2 I'm slightly more confused as I think I clearly see binding in the neutravidin only channel - but I don't see that same negative binding response in the neutravidin/PoI channel.
All sensorgrams/binding plots shown are buffer blank subtracted, where 1 % DMSO buffer (to match the 1 % DMSO content of all compound-containing injections) has been injected over the same surfaces.
Key questions:
1.) Why do we think I'm not seeing the negative binding responses for Compounds 1 and 2?
2.) Why am I not seeing the same binding responses/sensorgram profile between Channel 6's Fc2 and Channel 7's Fc1 (both should be neutravidin-only).
Many thanks for any thoughts and help!
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- Arnoud
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3 months 2 weeks ago #2
by Arnoud
Replied by Arnoud on topic Negative Sensorgrams
High, sorry for the late response.
I think you are right but did you try the the volume exclusion correction for your compound 1 and 2?
Since matching DMSO concentration can be challenging.
Maybe interesting to read www.sprpages.nl/troubleshooting/negative-curves
P.S. the slides are too small wit too much information to be readable. Please upload one sensorgram at the time.
kind regards
Arnoud
I think you are right but did you try the the volume exclusion correction for your compound 1 and 2?
Since matching DMSO concentration can be challenging.
Maybe interesting to read www.sprpages.nl/troubleshooting/negative-curves
P.S. the slides are too small wit too much information to be readable. Please upload one sensorgram at the time.
kind regards
Arnoud
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