I performed 2DE from cancer cell line. I had 4 gels, one with control, other are treated with anti-cancer drug with 3 different incubation time but I could not find any differences between the four gel. What could be the reasons? Might it be the concentration of the drug or the incubation? or something in treated the cells?
I suppose you are looking for some expression differences.
What is the drug and what is the target?
Do you find the same level of target in all 4 gels?
Did you try any mass spec base quali/quanti analysis?
Moreover, it would be helpful if you disclose more details about your protocol.
I cannot discuss any of your question, for I do not know the concentration of the drug you used, the incubation time, the type and number of cells, the medium, the temperature etc etc.
Any of these factors can be addressed as a problem.
Their are a number of reasons:
(1) The optimal Drug concentration is ineffective at high-number of cells, prepared for 2DE. The drug is most effective at lower-cell number. Therefore, apply the drug to lower cell numbers and collect multiple dishes.
(2) Maybe you have small changes, in the shift, but your staining is not enough to show all changes.
(3) Probably not all changes contribute into migration shifts.
(4) Probably, the drug affect on expression/degradation pattern of proteins, but 2DE is not appropriate for spot-inensity-differences.
Good Luck ......
Beston
There are a number of possibilities: one could be the drug concentration, another could be the time of incubation. It is also possible that the medium of cell culture is not permitting drug entry into the cell. Another possibility could be that the mode of action of the drug is not by directly affecting the cancer cells but by modulating other factors/cells.
Hi..If you can share experimental details, like which stain did you use..incubation time..drug concentration etc..would be good to suggest you..still
I would suggest you to run SDS-PAGE to atleast compare the profile on 1D before proceeding for 2-DE.
Instead of 2DE if you are able to perform mass spectrometry based quantification, would be good to access expression changes since 2DE has its own limitations..
Keeping in mind what Beston wrote, what kind of change do you expect?
What I mean to say is: your drug should change the expression (or the level, at least) of several proteins, if it is working.
Obviously, it could be that with your naked eyes you cannot really see the difference.
I would suggest you scan your gel and use a software to evaluate the differences.
If even this approach fails, you can try to an in-gel digestion and LC-MS. That will pretty much give you a final answer.
Please do consider that, by performing in-gel digestion + LC-MS, you include several other factors that need to be evaluated in order to get good confidence in the end result.
A final consideration: I would start from a much higher drug concentration and than decrease. At least you can get a gel with a "sure result" and compare it with those obtained with lower concentrations of the drug,
Though I suspect you will have some "strange results" when comparing high and low doses for this particular drug.
Hi Eric,
You had used 3 nM of bortezomib on myeoloma cell line. May I know how did you decide to use this concentration. I would suggest you to first determine the IC50 concentration of this drug for your cell line then you start with that concentration. Later you can see the effect of increase drug concentration as well. As Fabrizio and I also suggested earlier, in addition to 2DE also perform LC-MS/MS to see the changes in expression level. Good luck. Thanks....
- Badges
- Science topic