hello all,
I am trying to immobilize oxytocin on a CM5 chip, @200ug/ml concentration, and pH 4.0 acetate buffer.
Oxytocin is a 9 amino acid long peptide with a sequence "CYIQNCPLG", the first and sixth cysteine makes a disulfide bond.
I am not getting good immobilization at all.
I am hoping EDC/NHS was good and its something to do with the lack of lysine in OXYTOCIN
can anyone suggest me other ways of immobilization that I can use for oxytocin immobilization?
Thank You
You have no lysines but you still have the amine from the first cysteine. How accessible it is for reaction with COOH I cannot be sure. I assume that your reaction conditions are not ideal. Indeed, acetate buffer at pH 4 is concerning, as the acetate COOH groups could react with the EDC. pH 4 is also a bit too low, try MES buffer, pH 6. If you cannot get it to work with different conditions, you could purchase a BSA-oxytocin conjugate (probably made using EDC) and conjugate through some of the free amines left on the BSA.
I appreciate your suggestions, Nick Gee.
We typically utilize pH less than the pI of Protein for maximal immobilization since dextran (on-chip, PI of 3.6) and protein will then have a higher electrostatic interaction.
I chose pH 4 because, according to the protparam, oxytocin has a PI of 5.5.
The BSA-oxytocin idea seems like a good one, and I would try it.
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