Hello everyone,
I would like to observe the effects of a recombinant protein on in vitro cultured cells. For this reason, I need to know a concentration at which recombinant proteins can be used in in vitro culture without killing the cells. I've been looking for information but I couldn't find a proper answer to my question.
Did anyone ever work with recombinant proteins incubated with cells in vitro?
If so, does anyone have any tips about working concentrations, at least a suggested starting work concentration, from which I can start?
Thank you in advance to you all!
Have a nice day
Using bacteria you might start somewhere between 1-10 mg/ml (usually somewhere between 100-1000 uM). It all depends on your yield and how toxic the protein is to the cells. Do serial dilutions down to nano/picomolar concentrations to find where you should be. You be be able to use lower concentrations as you probably have fewer cells if you're not using bacteria.
To answer that, we would need to know what your protein is, and what it's supposed to do. For example, we use Albumin to carry nutrients to the cells at up to 10 mg/ml. We use a cytokine like IL6, to activate its receptor, as low as 10 pg/ml. So that's potentially a very broad range. Feel free to tell us some more, and we can probably narrow it down a lot.
Finally another thing to pay attention to is toxic contaminants in your protein preparation. For example, if your protein was made in E. coli, you're going to have some level of endotoxin contamination that can kill the cells or interfere with your assay. You want to measure that, and understand how much endotoxin you're putting on to the cells with your protein, and what the effects of that endotoxin might be.
There are multiple things like that, which can all ruin your day. It all depends on the details.
I worked quite a lot with rh and rm proteins in vitro. The answers depend on the source of the protein if you are purchasing the recombinant protein make sure that it is carrier-free or at least that the carrier not harmful to the cells. Many cheap rh proteins sold as control proteins (i.e. ELISA, WB) are usually not useful for cell culture application due to preservatives such as sodium azide. Then as suggested by Simon Caulton you need to determine an effective and non-toxic concentration of your protein to the target cells. Another important question is how the cells are supposed to interact with the target protein. In the case of the cell membrane proteins with the known receptors is fairly easy to get meaningful effects by simply adding the protein to the cell culture media. For many proteins that are located within the cytoplasm or nuclei, however, as well as proteins that are subject of activation or posttranslational modifications this approach is usually not effective and overexpression by plasmids is preferable. The direct delivery of the protein to the target cells by lipofectamine is possible, but usually not effective due to the harmful effects of the transfection process to the cells that usually mask the effects of the target protein.
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology
- Culture Cells