Posted by The bane of my existence for some time now.
I dont talk much about my research here, but its safe to say I consider myself an inventor who works in virology. People say “Damn. Its so hard to do ___. I wish there was an easier way…” and Im like “… I can do that.”
Gimme a plasmid, a primer (just need one), a paperclip, and a package of ‘Fruit Stripe’ gum, and I can make your retroviral dreams come true.
I make stuff, and then pass it off to other people to like, do research with it.
So I made myself a couple new toys. I then have to characterize my toys so other researchers know how to use them (basically write the product stats and instruction manual). The ‘instruction manual’ has been done for months, but there was one thing holding the publication up.
Nef.
Nef is not a ‘core’ retroviral component (gag, pol, env, LTRs)– its an ‘accessory’ HIV-1 (and some of its relatives) has. Has some neat features from the perspective of a virus and from the perspective of evolution as a whole. But like, I fidgeted with it a little bit, and I really thought it should have been there… Yeah…
I couldnt find it ANYWHERE.
HOW DID I LOSE NEF???
Well, first of all, its hard to *find* Nef in the first place. Nef is this teeny tiny protein. Its not an enzyme, so you cant test for its presence with some kind of simple assay (substrate + enzyme? = yes or no). It doesnt glow. It pulls cellular proteins off the surface of infected cells, but its kinda hard to really ‘see’ that in an assay, and then there are other HIV-1 proteins that do the same thing. Even if you saw an ‘effect’ of down-regulation in infected cells, you couldnt be sure it was BECAUSE of Nef. It wouldnt be as cut-and-dry as say, a reverse transcriptase activity assay (either RT is there and reverse trascription occurred, or it isnt and nothing happened).
What I needed were antibodies to Nef.
Antibodies with enzymes glued to them, that were specific for Nef proteins, could be used to detect the Nef protein in a Western Blot. Or, my go-to-protocol– make everything radioactive in a cell with radioactive sulfur (35S. every methionine, the start of every protein, will have a radioactive sulfur, with means all of your proteins will be ‘hot’. cysteines are included too, but methionine is the big one). Anyway, when all your proteins are ‘hot’, you can use antibodies to Nef to purify the Nef protein, run it out in a gel, see a radioactive band the size of the protein you are interested in, YAY! Its called an immunoprecipitation. I mean seriously, this is like *my* thing– when in doubt, make it radioactive! YAAAAY!
Minor problem: I have done approximately 5 million Western Blots and 10 million IPs.
No Nef.
Why?
CAUSE MY ANTIBODIES WERE CRAPPY.
:-/
If your antibodies dont see the protein, you dont see the protein.
I ordered antibodies from EVERYWHERE. EVERYWHERE. They didnt WORK. So finally I did what I should have done right off the bat– contact my go-to-reagent company.
Me: Hey, hey man. Can you hook me up with some anti-Nef?
Tech Dude: Yeah! We got that.
Me: What kind you got?
Tech Dude: We got some hyperimmune sera. Rabbits.
Me: *shudder with ecstasy* Does it work on IPs?
Tech Dude: I dont know about that. But it works in Westerns 1:8000 primary. And in ELISAs.
Me: 1:8000 PRIMARY?? *shudders again* Hey, I dont suppose you could hook me up with some of your immunogen for a positive control. I just need a little, man. Common.
Tech Dude: Sorry, we dont sell that. Its just in house.
Me: *shamelessly begging for 45 minutes*
See, the antibody was just one side of My Problem. For all I knew, the antibodies I was using were fine, but my timing with the virus was off. I had a ‘positive control’ for Nef– just a regular ol lab strain of HIV-1. But Nef is only made very early in infection, and then HIV-1 moves on to other proteins. If my timing was off, if I was too late, I would never see Nef, even if my antibodies were fine.
I needed some pure, free base Nef for a positive control…
So I had to make that. Amplify the nef gene, plop it into an expression vector– a DNA sequence that you can put into cells that just screams “GO!! GO!! GO!! GO!! GO!! GO!! MAKE NEF!!!!”
But how do you know you made Nef from that unless you have an antibody that you know works??? Well, then I had to put a little tag on the Nef that could be seen by an antibody I know works. So if I could see Nef with the tag antibody, but I couldnt see it with the Nef antibody, then I knew the protein was fine, but the antibody sucked. And if I didnt see either one, the protein sucked.
So I got THAT to finally work.
So I finally had ALLLL the controls I needed to do the one experiment I needed.
And Nef wasnt there in my new toy.
I mean, thats okay, its just something I need to tell everyone in the instruction manual. But all that time. All that effort. All that frustration… and Nef was never there.
I told one of the undergrads that this had to be an analogy for something.
And then it hit me:
Jesus.
Nef is like my Jesus. People pouring their hearts and souls out to him, their time and money into him, desperate for him, and he was never there.
LOL!
Or its like Kenny.
Labmate: “YOU KILLED NEF! YOU BASTARD!”
Me: “IT WAS A BASTARD TOO!”
Ugh.
But I definitely killed it. Now I can move forward.
July 6, 2011
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