I am considering using a Luminex based assay to measure protein expression. Does anyone have a way to normalize for variability in cell density/well?
Welcome on board ScientisSolutions,
Sorry i didnt answer you earlier.
The way i am doing it is but nrmelizing to total protein concentration.
Thanks for your response. I will be measuring sups from attached cells, and would prefer not to detach the cells until after I get the results of quantitiation, so I would be looking for some secreted "housekeeping-like" gene that I could use to get an idea of cell mass.
Still, after you will finish you assay, you can wash the plate twice with PBS. Then add some RIPA buffer for 10 min on ice. After that you can do a protein concentration assay. We use BCA.
I will be measuring sups from attached cells, and would prefer not to detach the cells until after I get the results of quantitiation, so I would be looking for some secreted "housekeeping-like" gene that I could use to get an idea of cell mass.
The "housekeeping" protein you are looking for may be problematic. Your choice will be dependent upon your cell type, since there is no one protein that is constitutively secreted by every cell, and determining which proteins are constitutively secreted by your cell type may be difficult.
I think you have two options:
1) Those who use ELISA for detecting proteins in cell or tissue lysates will often express their results as mg protein/mg cell mass. In your case, this would essentially measure the amount of protein secreted per cell.
2) Look for your own "housekeeping" proteins in your sample data. No matter which set of proteins you are detecting, there are bound to be several that will change very little in concentration under your experimental conditions. Once you have identified those, you can compare the relative concentrations of other proteins to that "housekeeping" protein.
Or, if the cell density is highly variable, you can look at the ratios of the relative protein expression. You will probably see some kind of pattern...the absolute concentrations may change slightly for a set of proteins, but the relative ratios of these will not. These are your "housekeeping" proteins. You can then use these for normalization of your results.
Notice that neither of these methods is truly measuring the "concentration" in solution, only the relative amount of protein secreted. If this is what you need rather than the absolute concentrations, you could use an antibody array. These assays use multiple capture antibodies spotted onto a grid pattern on a solid surface to measure relative differences in protein expression levels, not just yes or no. It detects many proteins in the low pg/ml range, and you can measure with changes in expression of even 1.5-fold with good accuracy.
This would probably be cheaper than using Luminex, and if you use a membrane-based array, you wouldn't need a flow cytometer, just some type of chemiluminescence detection, the same as for a Western.
What kind of secreted proteins are you trying to detect?