Blocking step

Dear All,
good day! Hope you're doing well. I want to clarify one thing in blocking step. If there is anyone who worked on the CM5 chip and was blocked by biotin. Biotin is available in powder form and is recommended to be solubilized in ammonium hydroxide. How did you use biotin? Are you diluting in running buffer or injecting a solubilized solution? Thank you.

Hi,

Do you mean the blocking step during immobilization? And if so why do you use biotin?
Can you give some more context?

Arnoud

Dear Arnoud,
hello. Thank you for your reply. I need the blocking step to avoid non-specific binding. I'm using biotin, to make streptavidin biotin interaction. Yes, I mean the blocking step during immobilization (After how I immobilize biotinylated aptamer, before the protein injection). Free streptavidin needs to be blocked, right? Thank you.

Zhazira

Dear Zhazira,
You can make a 50 mg/mL biotin solution in 2M ammonium hydroxide. Then dilute to 25 µg/mL in flow buffer (you may adjust the pH to around 7.4 if necessary) and inject to block free streptavidin sites.
If there is no non-specific binding by your protein you don't need to block the streptavidin. But the only way to know is to test.

Kind regards
Arnoud