Abnormal +4000 RU Baseline After EDC/NHS Activation with No Ligand Immobilizatio

Here is my current situation:After EDC/NHS activation, the RU does not return close to baseline. Instead, after washing it stabilizes around baseline +4000 RU, whereas tutorials show it should typically remain around baseline +200–300 RU. During ligand injection, I also do not observe the typical gradual RU increase — the signal remains almost flat. However, my preconcentration test looked normal.Reagent details:

• EDC: 750 mg dissolved in 10 mL deionized water (self-prepared)
• NHS: 115 mg dissolved in 10 mL deionized water (self-prepared)
• Ligand diluted in sodium acetate buffer, pH 5.0
• Ethanolamine prepared from pure reagent and adjusted to pH 8.5 with HCl

My main questions are:

1. After EDC/NHS activation, what RU level is normally expected after washing?
2. Is it acceptable to prepare EDC and NHS stocks manually in DI water like this, or could that be causing abnormal activation behavior?
3. Has anyone seen a similar baseline behavior after EDC/NHS activation? Any suggestions on likely causes or troubleshooting steps would be greatly appreciated.
4. Could this abnormal high baseline (+4000 RU) be caused by hydrolysis of EDC and formation of side products (e.g., urea derivatives) that accumulate or adsorb on the surface?[img]IMG_1303.jpeg IMG_1301.jpeg IMG_1302.jpeg[/img]

Short answers:
1): normally between 125 - 250 RU on a dextran based surface (CM5)
2): Yes, I dissolve a full bottle and then aliquot in single activations (150 uL) and freeze at -20 celcius for up to 2 years.
3): Not the high baseline after immobilization.
4): I would say no, and they will be washed away by the deactivation.

Unfortunaly the images where not uploaded. Can you try again?

regards
Arnoud