Low Intensity Signal

Hi Arnoud,

I have been running some very basic binding assays to test the performance of the machine. Specifically, BSA vs anti-BSA and HSA vs. Anti-HSA. In both cases the intensity is extremely low.

Upon looking at the raw data, I discovered that the reference channel is increasing as the concentration of analyte increases. So that when the reference is subtracted from the other channels, the final intensity is low. Do you know what could be causing this?

Thanks a lot

Hi,
Depending on your reference channel (unmodified, deactivated or non-related protein) some non-specific binding can be expected. What is your reference made of? This depend on the analyte. The antibodies are pure?
Some extra non-specific binding with higher analyte concentrations is normal but your specific response should go up faster. Is the binding curve normal 1:1?

Kind regards
Arnoud

The reference has been unmodified. I have been using a mixed peg/cooh chip. And yes the antibodies are pure.

The binding curve does not look normal at 1:1 and what I have observed is the reference increasing at nearly the same rate as the specific response.

The fact that the reference is increasing at the same rate as rhe specific response can be a sign of non-specific binding. I would suggest to deactivate the COOH groups (e.g. with ethanolamine) of the reference channel to have less charges on this channel.

Arnoud

I block the reference with 1 M ethanolamine-HCl pH 8.5. Is there a reason why this would not be effective?

Thanks for your help.

The activation-deactivation step will probably not convert all COOH groups. Depending on the activation time a smaller or larger fraction of -COOH groups will remain. I don't have absolute values for that.