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Non-specific binding or real binding?

  • labrats
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5 months 4 weeks ago #1 by labrats
Non-specific binding or real binding? was created by labrats
Hi everyone,

This is the SPR test graph we have obtained from collaborator, aiming to validate the bio-chem active compound from HTS. Unfortunately, the collaborator told us the compound is "non-specific" (see pic attached) binding.

We are wondering if you know and can help us? Thanks in advance!

The experimental setup:

Instrument: Biacore 8K+
Chip type: (CM5 chip)
Immobilized ligand : (His tag in N-ter /82.2 kDa)
Immobilization type : (Amine coupling, NHS)
Immobilization buffer: (10mM NaAc pH4.0)
Running buffer : (20mM Tris pH 7,5 NaCl 150mM, 0,05% tween, 5%DMSO)
 
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  • Arnoud
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5 months 3 weeks ago #2 by Arnoud
Replied by Arnoud on topic Non-specific binding or real binding?
Hi,
First of all, the steady state graphs are wrong since the kinetic curves below are not going to steady state. Therefore: don’t look at them. Assuming that all three sensorgrams are about the same compound, the main problem is, that there is no saturation (colours in the steady state graph don’t seem to correspond with the colours in the sensorgram) . Between the experiments the outcome looks not consistent (compare Rmax figure 3 and 4).
I agree with the result “non-specific” binding.

Kind regards
Arnoud
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