I am currently carrying out work analyzing the kinetics of progesterone receptor/ligand interactions using SPR. Currently when capturing the progesterone receptor (PR), the running buffer does not contain DDT, and capture can still be performed normally.
However, we are not having great success in identifying the interaction between ligand and receptor, with non-specific binding being a large problem in the reference channel. In similar experiments that use the estrogen receptor rather than PR (Rich et al. 2002), DDT was included in the running buffer.
I would like to know what the significance of adding DDT is to the running buffer. All other conditions that we use are the same as in the Rich et al. publication, apart from the immobilized antibody: we use anti-GST and a GST-tagged receptor, whereas they use His4mAb and a His-tagged receptor.
I have attached the publication for convenience.
Hi,
be careful, they added DTT and not DDT!!
DTT is a reducing agent and can be included into different buffers to prevent protein aggregation (it prevents intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins). If it's concentration is too high it can be detrimental for the protein folding, but not at 1 mM concentration, as reported by Rich et al.
Try to add it for your SPR experiments, even if I don't know if it can interfere with your immobilization system (GST-tag/anti-GST Ab).
Good luck
Ah thank you! That was a typo, I won't be using a banned pesticide! Thanks for the advice.
James
It would help to know the actual SPR chip configuration that you are using: are you relying on bare gold and a non-specific interaction, or one of the many paradigms for immobilization?
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