I am working with stem cells stably transfected to express the neuropeptide PACAP. We controlled the expression of the transgene by RT-PCR and the presence of the protein in the cells by western blot.
However, I would like to verify if the cells deliver the neuropeptide, so I would like to detect it in the supernanent of my cultures.
Does anyone know a good technique to detect small amount of a molecule in a culture medium? We already tried by MALDI-TOF but my medium is colored and avoids any detection.
Why don't you try to concentrate supernatant and carry out a western blot?
We are also trying to do that...It doesn't seem to work very well... I think that the problem is the very small amount of peptid that we expect in the supernatant... We have to load a very big quantity of protein to see something, that avoid a good migration... So we are not able to separate our peptid which is quite small (4kDa)..
Well, if u have an antibody for wb, maybe it also works for IP, you can try to IP supernatant and then wb
Have you tried Tris-Tricine SDS-PAGE?
It is difficult to separate small peptide by normal Tris-Glycine SDS-PAGE but you can clearly separate small peptides (2-4 kDa) with Tris-Tricine SDS-PAGE.
You may detect PACAP by Western blotting.
We have detected amyloid beta peptides (42 amino acids, 4.5 kDa) and humanin peptides (24 amino acids, 2.65 kDa).
http://depts.washington.edu/bakerpg/protocols/tris_tricine_gels.html
http://www.bio-rad.com/prd/en/US/LSR/PDP/L9KNGJ15/Mini-PROTEANreg_Tris-Tricine_Precast_Gels
Thank you very much for your answers !! The BIAcore system seems to be very interesting because of the high sensibility of the system. But, in a first time, i will try to use IP before analysing the proteins by western blot .
Thanks a lot.
The current HPLC-MS protocol is not very sensitive. A collaborator with a former lab of mine used tricine gel to some success. I developed an ELISA method, but it is not as sensitive as the tricine-WB method.
The best way is concentrate your culture medium with Millipore amicon ultracentrifugal concentrate filter....you can choose according to size of your protein and then you can do western blot on this concentrate medium by adding sample reducing agent. i hope it will be helpful to you....
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