Ligand Thiol Coupling

Hello, 
I want to immobilize my peptide with a free sulfhydryl group on CM5 chip. I used PDEA with 80mM concentration and immobilized 600ug/ml of peptide on the chip. As you can see in the attached figure, the signal after adding PDEA was 52 RU and did not change much. After adding the peptide, I got 162.9 response units, which was insufficient for my further analysis of analytes. When I increased the PDEA concentration to 320mM, the signal increased to 156, but after adding the peptide, the signal was almost the same as last time (137RU). What is the best concentration of PDEA and peptide, flow rate, or contact time to increase the amount of peptide on the surface?  
Thank you for your help.

Hi,
You don't say that you activate the sensor surface with NHS/EDC. Did you?
The recommended immobilization protocol can be found at www.sprpages.nl/immobilization/immobilization-procedures/thiol

I would use a less concentrated peptide solution (20-100 µg/mL). You can 'play' a little bit with the pH of the ligand solution. It seems that the pre-concentration speed is a little low. You can calculate the peptide pI and compare if pre-concentration is feasible.
As a last resort, can you confirm the -SH group on your peptide?

Kind regards
Arnoud

Thank you so much for your response.
Yes, I activated the surface with EDC/NHS, followed by PDEA. Afterward, my peptide was added, and lastly cysteine/NaCl. I am using synthesized peptide with cysteine at C-term. The PI of my peptide is 5.83, and it was diluted in Na-Formate with a PH of 4.3 to make it positively charged.
By lowering the concentration of the peptide, should I increase the contact time?
Would you mind explaining what you mean by pre-concentration?

Thanks again

For the pre-concentration look at www.sprpages.nl/immobilization/ligand-pre-concentration

We adding the peptide to surface you should calculate first how much you need to get a proper signal with your analyte. Please read the Immoblization section ( www.sprpages.nl/immobilization ) on the sprpages.
When it is possible to do the immobilization 'by hand' you can adjust the ligand incubation time until sufficient peptide is immoblized. We take the difference between before and after ligand injection and repeat ligand injection if we want more before deactivation of the surface (hope this is clear).

Arnoud

Thank you so much for the links and for the great help.
Great, I'll try it and see how it goes.

Thanks again,
Mehrnaz

I tried various buffers with various PH but they resulted in a negative/inverted response during pH scouting. The pI of my peptide is 5.83 with 1.2 KDa molecular weight. I used 600ug/ml for each screening with 420 second contact time. I also used HBS buffer as running buffer. My peptide was lyophilized in PBS. Is there any possibility that the PBS may change the PH and lead to this? Could you please help me how to fix this?
Thanks again,
Mehrnaz