Trying to detect an endogenous neuropeptide at very low concentrations (basal pM concentration, about 1ug total peptide in a single rat brain). Antibodies are not an option. Has 1 disulfide bond.
Particularly looking for an appropriate lysate buffer for homogenisation, how to get rid of proteins and salts, and trying to do as few steps possible so I don't lose my peptide somewhere on the way.
Dear Markus, You have asked a tough question. I must admit that I do not have a precise protocol for something like that, but I will give it a try. Maybe we can build on it.
So, 1. Do you know if this Neuropeptide present in/secreted from Dense core vesicles? Or Synaptic vesicles?
If that is the case, there is a protocol from Reinharth Jahn's group for the isolation of synaptic vesicles. I dont think they have checked if they also purify dense core vesicles.
http://www.ncbi.nlm.nih.gov/pubmed/23619891
2. Do you also want to eventually be able to quantify the peptide? because then the protocol must be very robust and hence simple. But the previous link I sent, I think the protocol is very robust.
3. In terms of getting rid of proteins and salts, I think once you have your sample (maybe the synaptic vesicle sample/dense core vesicle sample), The best way would be to use FASP. Its fast and clean. If you know the size of the peptide, you can use an appropriate filter. (10kd instead of the standard 30kD used in FASP).
Hope it helps. Like i said, it helps to know a little bit as to where the peptide is located subcellularly in order to have a starting point.
Cheers,
Nikhil
Oh and since it has a disulphide bond, so it probably forms dimers/oligomers. So keep that in mind.
Secondly check if the peptide is present/enriched in a particular brain region, which you can use for isolation.
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