The normal cell still lives when G418 is added, what is the lowest concentration of G418 to kill the normal cells but not the transfected cells? How can cells be made to express proteins at the same level?
What is the cell line you used and for how long did you treat them with G418?
Sensitivity to G418 or any other antibiotic is very cell line dependent, although they usually respond on the same dose range. The optimal concentration is to be tested experimentally. I suggest you to try different doses of G418 (between 50 to 1000 ug/ml) in a simple titration experiment using non-transfected cells, for at least one week. Since you already know the concentration that doesn't kill your cells, that could be your lower one, and then increase it by two-fold steps. Thus, you should see complete cell death and the opposite, from higher to lower doses of the drug, respectively. Choose the dose that kills 80-90% of the cells in that time period and use it as the selection dose on the transfected, resistant cells. You can do this using microtiter plates, or larger format plates by means of MTT or counting cells, for example. Usually, just looking at the % of surviving cells under the microscope can give you an idea of the optimal dose.
For the second question, do you mean different cell lines with the same plasmid or the same cell line? You probably need to use the same amount of DNA and painstakingly isolate stably-expressing clones and test them for the levels of protein expression and choose those with similar amounts...
Hope this helps!
First question: First thing you should do is do a kill curve on your cells with G418 - start at a low concentration and do either a semi log curve (e.g. 0.1, 0.3, 1, 3 mg/ml) or, as suggested above, doubling doses (0.1, 0,2, 0.4, 0.8, 1.6 mg/ml). Ideally you want to select the dose that results in 70-80% cell death upwards within 48h. A couple of things that may help. Following transfection of the plasmid, I usually find it best to split the cells into the media with antibiotic - rather than merely switching the media. Secondly, it is VERY important not to let the cells get anywhere near confluence with G418 - as G418 resistance can be transferred from cell to cell on contact. If you don't want to risk this for polyclonal stable cell lines, then i would generate monoclonal stables but remember to use more than one monoclonal cell line for subsequent experiments.
For the second question: Not really sure what you mean here, but as noted above, generating monoclonals (which is painstaking) and looking for stable cells of similar expression would be one option. The other option, if your plasmid has, for example, a GFP tag on, would be to take polyclonal cell lines and use flow cytometry to 'enrich' your stable cells above the desired level of expression. Note, you can also do this generally if you continue to have issues with G418 selection. However, I would recommend taking 3-4 batches of selecting cells, enriching them, and pooling them together to avoid clonal issues for subsequent experiments.
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