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Spikes in the sensogram

  • Zubryte
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1 year 6 months ago #1 by Zubryte
Spikes in the sensogram was created by Zubryte
Hello,

 I have an ongoing issue with spikes in my sensogram when doing manual injection (I did try wizard as well and stil had jumps.)

Since I had an issue with injection of my protein (hCG, MW:36kDA) onto the EDC/NHS modified surface, I decided to step back and test the protein injection on unmodified CM5 chip (Instrument Biacore 3000). I also followed the advice from SPRpages. I tried different things on each flow cell. For all experiments I used PBS buffer.

FC1: Injection of 6.3, 12.5, 25, 50, 100 mIU hCG at 10uM/min flow rate, injection volume 50ul, dissociation - 300s. (This is my standard protocol)  [hCG-Test_fr10-50ul_Fc1_CM5]

FC2: Injection of 6.3, 12.5, 25, 50, 100 mIU hCG at 25uM/min flow rate, injection volume 100ul, dissociation - 300s. (I increased the flow rate since it was mentioned that low flow rate can result in more air bubbles). [hCG-Test_fr25-100ul_Fc2_CM5]

FC3: Before the injection of the protein, all protein solutions were centrifuged at 13000 rpm for 15 min. Injection of 6.3, 12.5, 25, 50, 100 mIU hCG at 10uM/min flow rate, injection volume 50ul, dissociation - 300s. [hCG-Test_fr10-50ul_Fc3_CM5]

FC3: Injection of 6.3, 12.5, 25 mIU hCG at 25uM/min flow rate, injection volume 50ul, dissociation - 600s. (Again tried to increase the flowrate to avoid the bubble formation. Also increased the dissociation time.) [hCG-Test_fr25-100ul_Fc3_CM5]

FC4: Injection of 0, 1.6, 3.1, 6.2, 12.5, 25, 50, 100 mM NaCl in running buffer PBS at 10uM/min flow rate, injection volume 100ul, dissociation - 600s. (This experiment was suggested in SPRpages to use as a control).

 [NaCl   -Test_fr10-100ul_Fc4_CM5]

None of these experiments ended in a clean sensogram. Doeas anyone know what am I doing wrong? Or maybe you have some ideas why this is happening?

Photos attached.

Thank You!

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1 year 6 months ago #2 by Arnoud
Replied by Arnoud on topic Spikes in the sensogram
The spikes seem to occur at the same position each injection. This can point to something in the injections system (needle, tubing, syringes or IFC). I suggest to clean the system with the Supedesorb procedure. Then take the injectionport an clean it under the tap. In addition clean the injection needle by hand. Check the connectors of the tubing are leak free. The do a prime and check while priming that the syringes are filled and emptied properly (no airbubbles and smooth up and down).
Filter and degass the running buffer prior to use and Perform the NaCl test again.
If the spikes still persist you may have to replace the IFC which will be not possible since it is out of service.

Kind regards
Arnoud

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