Looking for an in vivo non-radioactive labeling method for Drosophila larvae

[G-Rex]

The topic title pretty much states my purpose. I'm trying to gain an idea of the effects on total protein production the knockdown of a specific gene product has on Drosophila larvae. My professor would prefer if I could find a method that does not rely on radio-labeled amino acid incorporation so that I can perform the experiments without his supervision. However, I'm having trouble finding any such methods. Does anyone know of any labeling methods that might work for what I'm trying to do? As I noted in the topic, we're going to be measuring in vivo, so any in vitro methods won't work, and I don't think fluorescent antibody labeling is feasible either, as we're trying to measure total protein - not just specific ones.

[CharonY]

What precisely do you want to see? Do you want to monitor protein concentration while the larva is still alive (e.g. real-time monitoring?).

[G-Rex]

What precisely do you want to see? Do you want to monitor protein concentration while the larva is still alive (e.g. real-time monitoring?).

 

 

No. We're going to be looking at thin tissue samples. We just need a quantitative way to compare how much protein we have in our mutants versus the wildtype. We've measured the larval protein concentrations using a modification of the Lowry protein assay on a bulk basis (one larva would be too small a sample), so now we need something that can either corroborate or contradict the data we've gotten from that, and do so on an individual basis.

[CharonY]

In that case you can use in vitro methods. If the total content is what you are looking for you could try to measure it with fluorescent dyes, which have a lower detection limit. Most likely you would have to use up a whole larva to get reproducible results, though. Theoretically you could also use these stains on tissue directly, however reliable quantification is really difficult. In most cases I would say that the preparation noise is much higher rendering it pretty useless for a quantitative assay.

[G-Rex]

Well alright. I'll take that into consideration. Thanks for the advice.

[DctrZaius]

So you have a mutant of interest but you're not looking specifically at the gene product that makes it a mutant, you want to look at the impact on all proteins in the organism? It sounds like you would have to do a massive proteomics study to achieve that. What about fluorescently labelled amino acids? What will measuring this result actually tell you though? Is it a gene involved in translation or proteosome function or something?

[CharonY]

From what I understood the goal was to figure whether total expression levels have changed (which would be indicative of e.g. reduced activity/growth) rather than the occurrence and expression of individual proteins, which could be resolved in a proteome experiment.