I'm starting to appreciate why so few labs do proteomics (well). When things go well, it's 1-2 weeks of work for often extremely useful data that can turn a year of measuring different things based on things the literature suggested to look at to a month of validating results on things no one would have bothered to investigate otherwise. After ~1 year, I have pretty much every protocol committed to memory, and I only occasionally have to look up exact volumes / concentrations. When things go badly, it's months of wasted effort and up to thousands of dollars wasted in reagents alone, not including salary or instrument leasing costs. At this point, two post-docs, a tech, and I have switched out most of our reagents, tried new antibodies, and still the data coming out of each of our runs has been crap. It's a real crowbar in the bike wheels and has thrown off our entire lab's schedule for close to a month now. In the mean time I'm catching up on my readings and trying to take my (one) class seriously. I have the growing suspicion that one of the professors may be the sort of person to take pleasure in ripping apart other labs' works. But I think it only makes me respect them more as I see how much crap makes it into even the top-tier journals of neuroscience. In other news, I climbed my first V3 on Monday! It's nice to see some progression, since I'd definitely hit a plateau since coming back in January. Still have yet to move my body in any pattern even remotely resembling a dance though.
While my proteomics are a little less crazy than yours (only a surface biotinylation assay) I can relate. I always have the feeling the proteomic methods (specially SDS-PAGE) are the least advanced compared to all the DNA/RNA stuff. Of course, this has a lot to do with the instability of proteins and handling but still, it is annoying. Messed up a PCR? no problem, doing another one is fast. Messed up a western? Oh, you don't have enough material left for another one? You had to sacrifice two mice to get enough material for one? The next batch of suitable mice is in 5 weeks? fuck this shit. I was recently invited to try bouldering. Maybe I should try it out. Actually, my room has loft bed with bouldering stuff (no idea how they are called) attached to it. The guy who lived here before used to boulder/climb.
Most certainly. Until you get true peptide sequencing (a la nanopore?) / some method of amplification, there will be huge sensitivity limits to proteomics that will never bring it to the single-cell level. Supposedly new multiplexed staining methods should help with that, but antibody-based readouts have their own set of issues... I'm hopeful for advances in that area next few years though. The mice I've been using take close to four months, plus however many days of gene induction, plus the wait time for this breed, which has been quite a while now. Definitely makes me nervous in picking which assays I run. I've been stuck on whether to throw my remaining sample for a kinase-activation assay, or to save it for more targeted blots... Bouldering is great though! After the first few rounds of forearm death, progress happens pretty quickly. I've been going since September and seen a grade increase ~every two months.I always have the feeling the proteomic methods (specially SDS-PAGE) are the least advanced compared to all the DNA/RNA stuff. Of course, this has a lot to do with the instability of proteins and handling but still, it is annoying.
The next batch of suitable mice is in 5 weeks? fuck this shit.