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Continuous response increase in Baseline
- Madhu
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3 weeks 2 days ago #1
by Madhu
Continuous response increase in Baseline was created by Madhu
I have been using His-tag capture chemistry to check his-tagged VEGF binding to Antibody. After every cycle the capture baseline keeps on increasing upto 200 ru specially on channel 2 (where the capture goes). I have quite strong regeneration of glycine pH 1.5 (2 cycles) but still I see some deposition on the chip surface which gives me inconsistent KD values for two different runs . I have tried to reverse the analyte and ligand combination by using protein A chip but still same issue. Could anyone suggest how to overcome this ?
Instrument :t200
Chip: CM5 sensor S series
Temp:37
Flow :50 microliter
Instrument :t200
Chip: CM5 sensor S series
Temp:37
Flow :50 microliter
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- Arnoud
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2 weeks 5 days ago #2
by Arnoud
Replied by Arnoud on topic Continuous response increase in Baseline
As I understand you have a NTA-like surface and capture a his-tagged protein (ligand) and have an antibody as analyte. The regenration with glycine induces a strong baseline drift. And the other set-up is to capture the IgG on a proteinA sensor and detect the His-VEGF. both have the same issue so we rule out the sensor surfaces (or we can't do anything to it).
As for regeneration the first cycle starts without drift and after the regeneration the drift starts? if this is the case i would explore some different regeneration solutions, also since your glycine pH 1.5 looks insufficient.
I recommend reading www.sprpages.nl/kinetics/regeneration and try the cocktail regeneration method. It will take some effort.
kind regards
Arnoud
As for regeneration the first cycle starts without drift and after the regeneration the drift starts? if this is the case i would explore some different regeneration solutions, also since your glycine pH 1.5 looks insufficient.
I recommend reading www.sprpages.nl/kinetics/regeneration and try the cocktail regeneration method. It will take some effort.
kind regards
Arnoud
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