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2 Questions: Functional Ligand and Reproducibility
- mtarca
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5 years 4 months ago #1
by mtarca
2 Questions: Functional Ligand and Reproducibility was created by mtarca
Dear Arnoud,
I have 2 questions.
1) The first one is in regards to calculation of functional ligand. I saw Biacalculations information in the site and followed the formula for functional ligand. I got about 163%. See attached. Is this typical? Why above 100%? The mol. wt. of the analyte is 20813. What should I expect for higher molecular weight analytes like 67kDa?
2) The second one is in regards to reproducibility of the KD's, if I run the same samples back to back (ie. different days). Is getting approximately 2x difference acceptable in the world of SPR? Also, see attached data.
Thanks.
I have 2 questions.
1) The first one is in regards to calculation of functional ligand. I saw Biacalculations information in the site and followed the formula for functional ligand. I got about 163%. See attached. Is this typical? Why above 100%? The mol. wt. of the analyte is 20813. What should I expect for higher molecular weight analytes like 67kDa?
2) The second one is in regards to reproducibility of the KD's, if I run the same samples back to back (ie. different days). Is getting approximately 2x difference acceptable in the world of SPR? Also, see attached data.
Thanks.
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- Arnoud
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5 years 4 months ago - 5 years 4 months ago #2
by Arnoud
Replied by Arnoud on topic 2 Questions: Functional Ligand and Reproducibility
Hi,
I was wondering if your ligand is an antibody? Antibodies have two binding sites thus you have to multiply the Rligand with 2 and then you get a functional ligand percentage of ~81%.
High affinity interactions rely heavily on the dissociation part of the sensorgram. A slight drift can alter the results already. Thus I think you are pretty good here. If possible repeat a third time and try to get a CV below 20-30%.
Regards
Arnoud
I was wondering if your ligand is an antibody? Antibodies have two binding sites thus you have to multiply the Rligand with 2 and then you get a functional ligand percentage of ~81%.
High affinity interactions rely heavily on the dissociation part of the sensorgram. A slight drift can alter the results already. Thus I think you are pretty good here. If possible repeat a third time and try to get a CV below 20-30%.
Regards
Arnoud
Last edit: 5 years 4 months ago by Arnoud.
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