So I am really enjoying the New Grad Lab (NGL), partly because I've gone back to what I know well - fluorescence microscopy. This means, after learning how dissect the imaginal wing discs from larvae (not very difficult), I've been staining and imaging up a storm. We are interested in Protein X (that I was moderately involved with in old Lab) and the role it may or may not play in the blood brain barrier, or blood nerve barrier as its known in flies. The antibody I've been using to stain for protein X is for the vertebrete form of the protein but it seems to be cross reacting well. So far I've been trying to figure out the optimal working concentration which is not as easy with wing discs as it with cells. If I was using cells (as in cell culture) I would've collected a few dishes and set up a dilution series and had the working concentraion figured out in a week or two. Unforunately with wing discs I need ~ 20 discs / stain with each 20 disc dissection set taking about 2 hours. I don't know about you, but I can only spend about 4 hours staring down a stereoscope before I feel nausaus and my back and shoulders hurt from being hunched over. This combined with the time it takes to actually image and deconvolve image stacks = 2 months before we have a working concentration. I think I've found a good working dilution (by complete fluke!) and set up this week to run a SDS-PAGE so that I could do a western blot to confirm the specificity of the antibody. Do you how many days it has taken me to run the freaken protein gel? According to the date on my lab book I started this on wednesday. Three days later and a good number of attempts later the fucking gel has FINALLY set. Thankfully I have learned not to immediately start wondering WTF with me that I can't set a stupid gel to set. Though I would now like to say WTF. Its a bloody SDS-PAGE???? How difficult is it to mix together components!!!
Well apparently quite difficult when critical reagens like the acrlyamide and temed have gone off! This morning was spent making up small volumes of stacking gels to determine first if it was the temed (since we have like 6 bottles of temed we had to test them all), than if it was the acrylamide. We have determined the acrylamide is off for sure, so we've ordered more in. To make sure I don't fall to far behind, I bummed some acrlyamide off a neighboring lab to make up yet another gel. 3 days later I have an acrylamide gel sitting in the fridge waiting for me on monday morning. Lab work is 90% trouble shooting. Repeat. Repeat. Repeat
Posts
11 months ago
5 comments:
I read part your post and thought, "Cool, dissecting imaginal wing discs!" (I've never done it, just looked at pictures of them. I was not trained as a fly person).
Then I kept reading your post and went, "ugh, that's the bad part of lab work!"
And then... forgive me Scientistmother... but I couldn't help laughing a bit at the end. I've never had acrylamide go off on me, but I've had temed go off, and yeah, the protein gel WILL NEVER SET if the temed is off. You'll wait till Doomsday.
Troubleshooting, yup. Sometimes I miss it, a little bit. Most days I don't =)
Yay for troubleshooting ... erm ...fun. I think I'll have to pick your brains soon about fluorescent microscopy though. Man it's been ages, I never really did much, and now I'm supposed to teach people!
Yup! TEMED and acrylamide don't last very long in solution, particularly. Neither does APS--I sued to make it up fresh every time and make sure it crackled when I dissolved it.
NOWADAYS in my lab, we buy pre-cast Invitrogen gels, because those home-made gels are ridiculously irreproducible and we just got sick of it. As the boss, I think of everybody's time as per how much it costs me per hour. A grad student costs about $10-12/hr. A day of their time running, transferring and processing a gel for a western? $80-100. The two weeks of time they spent running the experiment and preparing the sample they ran on the gel? As much as $500-1000, not to mention all the resources that went into that experiment, and the antibodies they use on the Western, etc. An Invitrogen gel running-box? Free with a case of 5 boxes of pre-cast gels for ~$500 (50 gels).
Well worth the investment.
sued = used. I never sued my advisor for making me use it, but maybe I should have.
Bean-mom - nothing to forgive with regards to laughing. If I wasn't so frustrated I'd have laughed too. This is the first time I've had either the acrylamide or Temed. Usually making up new 10% APS fixes the not polymerizing issue.
Arlenna - I agree that it may be more cost-effective to subvert the troubleshooting process, but as frustrating as I find it, I strongly believe its an important component in the learning process. I would never have known that TEMED or acrylamide could go off if not for this experience. I am sure I have probably read that fact many times over, but without the experience it would not have stuck.
Mel - ask me anytime, I'm happy to help!
Post a Comment