I am shortly going to be doing a project where I analyze specific proteins that some insects secrete into the waxes that they biologically produce. I am interested in the quantity of the proteins they are submitting, but even more in the quantity that they are TRYING to secrete into these waxes. I am inclined to approach the method by studying how much mRNA for these proteins they are producing by PCR, as opposed to trying to assay for the proteins directly, using their activity. It is tolerably easy to isolate their secretion glands, and somewhat harder to purify the proteins out of the wax or to test their activity while they are embedded into the wax. I do have HPLC available as a technique, but it is tougher to do that when the enzymes are still wax-linked or wax-associated. It is also cheaper for me to approach it by PCR. Does anyone have any thoughts about whether I am right to take the mRNA approach to see how much they are elevating their production levels, or is this going to be a case of me beating my head against a wall, when directly assaying the enzyme activity or HPLC assaying for the enzymes might work better. Or is there another technique I haven't thought of that could tease out quantities of different enzymes embedded into a wax matrix?
Gas Chromatography for lipids and direct sequencing for proteins (Article Insect cuticular proteins
).
Why GC, M. I. Cooper? I use it to track small organic molecules in the wax, but would it work if I was looking at proteins? Some of the secreted proteins are really quite large. Would this technique work if the proteins are so big?
Do not use GC! I understand you preferring PCR but would recommend the harder way. There are too many steps in between( translation, PTM, secretion) and imagine the frustration when you finally performe the protein assay with a negative result! Good luck, Simon
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