|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
or tissue lysates
will often express their results as mg protein
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
, 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?