So class, I have a quick question about Co-IP detection. In the case of MULE, my antibody would bind EGFR (bound to MULE), then wash out unbound crap to have Ab-EGFR-MULE. Under normal circumstances, I would just run this on a gel and do a western blot against MULE to confirm binding, but without the MULE Ab this doesn't work. Is there another way of detecting secondary targets in Co-IP? otherwise I'm just thinking of doing a different binding assay, but I'm curious about how the MULE discoverers did it.
Also, someone talked about using RT-PCR from MULE RNA to isolate the gene. But given the similarities with RAS, wouldn't we end up with a mixture of the two? I'm thinking in terms of primers, because the only differences are in UTRs, which you won't have in the cDNA... so how can the primers be specific for just MULE?
Hey folks, I already emailed about what is "known" about MULE's gene, but there's another piece of advice I can share. Someone asked about the appropriate level of detail ... this will perhaps be the toughest part of the assignment to get "right", but it will be a VERY important thing to get accustomed to as we move forward. It will be similar to the Methods sections of your 367 lab reports, but even less. If, for instance, you wanted to propose knocking down a gene, you need more than to say, "I will knock down the gene" ... but less than, "I will add 4.6 microliters of such and such ..." I need to know the specific experimental strategy including variables and application-specific modifications, but don't need a thoroughly laid-out experimental protocol. I hope this makes sense. One of the things you'll need to include from time to time is the availability of certain reagents, kits, resins, etc. I made a list of vendor websites and put it in the Primary Literature Resources page. I think you'll find the information that these vendors provide to be helpful in constructing proposals. Good luck!
You may either email me your paper by this Wednesday or put it in the wiki yourself ...
Your mid-term exam will be on Monday, April 5th, during class. It will be a 50 minute exam consisting entirely of short answer questions. The material to be covered is everything before spring break (all reading and lecture materials). Expect questions to be things like: experimental descriptions, experimental differentiations, experimental mini-proposals, etc. Please let me know if you have any questions.
The reading assignments and overheads for this week are available on the Weekly Assignments page. Enjoy!