Can 1000 micro molar concentration of 63 kda recombinant protein used for cell line cultures or cell in studies
Hi there,
The question is actually : is 1mM within the biological range of concentrations for the protein considered? I'm afraid the question might be answered only if the protein is identified. What I can say is that 1mM is a lot for a circulating protein (as you want to treat cultured cells with your protein, I guess it is a secreted protein).
The most abundant circulating protein in plasma (not including the hemoglobin in red blood cells) that I know of is serum albumin, at a concentration in the 500 micromolar range. Everything else is at much lower concentrations. This paper shows the abundances of the most abundant serum proteins:
http://www.plasmaproteome.com/Papers/The%20Plasma%20Proteome%20Anderson%20and%20Anderson%20as%20printed%20corr1.pdf
Thank you for the answer yes it is the excreted /secreted protein found in filarial worms.
What the effective concentrations are in vivo compared to in vitro may be dramatically different, especially when working with surrogate cell lines or primary cells. The early stages of developing the drug Nplate is an example. In a primary or cell-line culture the secreted factor may experience many things that it does not experience in vivo due to the how, when, and where the protein is to target the interacting solute as well as the culture conditions and media (chemically defined or not). I'm guessing you might be working with the Brugia parasite which was of interest because of an earlier experience with research in trypanosomiasis. In a heterologous system the concentrations may have to be empirically derived but you might start with what is know in vivo from an infected host. Isn't that data available? Can you disclose if the protein is from the host or from the parasite?
Dear Grant thank you for your answer what u guess is correct the protein what i used is from Brugia malayi parasite protein.Plese can you suggest any article where they used 1 mM concentration of protein used for the cell culture assays
Now I see the earlier response regarding the provenance the 64 KD protein being of Brugia secretion. Are you then culturing lymphocyte cell lines and adding the recombinant protein version exogenously? Which lymphocyte class and species are involved, for example human T's of Jurkat lineage or murine B's of CH3/eB lineage? I'm not sure of the indication and therefore the cell lines you are using but I'm assuming this involves lymphatic filariasis or elephantiasis (skin). What will really help is knowing what type of response is expected from the cells in culture and the density of the culture, days of culturing, and protein stability.
If there's a receptor or some binding response, for example, then perhaps labelled protein can be used to do FACS and get a quantitative estimation of the amount of binding per cell which in in turn might give you an idea of how much protein to use in culture based on binding. On a functional basis, dose-titration may be the best practical solution, As a starting point for the amount of protein to use and going back to my earlier question about what is known in infected hosts, the CDC should have some data about the circulating levels of nematode antigens from both dermal and lymphatic infections using ELISA and other immunological methods. I assume there's polyclonal antisera to the 64 kD protein, right?
You might survey other parasitic infections such as toxoplasma to obtain an idea of the circulating antigen levels but not use the level of just any circulating blood protein to determine how much of the 64kD protein to use. Again, the concentration to use may not be representative of what happens in vivo and the in vitro concentration will likely have to be higher than values you find in vivo, maybe an order of magnitude or higher depending upon the response you want to observe.
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