Spikes with injection

Dear all,

I have some problems during the injection.

First, all the facts.
I'm using SA Chips (from Xantec) where small RNA is immobilized (~300 RU). I inject a protein where the RNA is binding to, and I want to determine the binding affinity at 37°C.
All my buffers are filtered, degassed and checked for RNase contamination.
I'm using a Biacore 3000.
I did this already successfully and now I want to reproduce it, and actually fail...

I immobilized a new Chip and did some test injections. They did not look perfect, but it seems to be on the right way.
Suddenly nothing worked. Directly at the injection start, there was a very intense spike and no binding was visible afterwards.I thought that the RNA is degraded after ~1 week at 37°C.
Therefore I immobilized a new chip.
The immobilization worked, but also here again just a spike is visible.

As I did this on different days, with different chips and with different aliqouts of the protein I can neglect air spikes.

Do you have any idea what the problem could be?

Thnx in advance!
Julia

Hi Julia,

My first move would be to validate the injection system by injecting flow buffer and a solution with some extra salt (+ 50 mM NaCl) to make sure the injection system is within specs.

If not, wash the system (desorb, sanitize if possible) and check if the injection port is clean and fixed properly.

Can you post a figure of the observed spikes?

Kind regards
Arnoud

Hi,

I always have a run just with buffer as reference.

Attached you can see the spikes.
On the left an overview. The first spike should be the injection and the second spikes are the regeneration (50 mM EDTA and 4 M Urea).

The picture on the right is a closer look on the injection side.

In red is an injection with the running buffer and in pink the injection with 100 nM protein.

Tomorrow I will follow your advise. Thnx

After cleaning with sanitize, the machine is working like a new one

Thank you for this very fast and helpful tip!

Glad to be of help.