| Hoyeta said: |
| 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?