No I'm not Dead

This is prompted by two emails. Both from good friends. Email #1 is from a friend who got Shingles, I think - (hope you get better, we'll all drink to your health Saturday during food-orgy ... I mean bookclub). The second email I received yesterday from a friend down in NYC asking me if I was still alive. It's good to know that I have at least two regular readers, although they are good friends first.

So what is happening?

I'm undergoing a severe case of last-experiment syndrome. (See this post, item #9).

Friday: Injected my brains out in the morning. In the afternoon, after intense discussion, I come to the conclusion: I need to perform this last experiment for the paper. I consult collaborators about obtaining some purified protein. They have some! I order a second injection marker (hope it gets here on time), plate the cells out and drink heavily at happy hour.

Saturday: Spend seven hours on the microscope imaging Friday's injections and collect the last of the data for the "background experiments" section of the paper. During lunch I flip through Science. I see another paper describing a silent mutation that affects the folding of the translated protein. Also, there is a cool paper on centrosomes and germ-line stem cells in Drosophila. Too bad flies can exist without centrosomes. I think about blogging but then the thought vanishes. Saturday night was spent with friends at Indian Cafe in Harvard square then at the American Repertory Theatre watching a rather good (and very modern) interpretation of Racine's Britannicus. Nero playing electric guitar, Junia videotaping herself in the midst of incest and backstabbing, Agrippina is fantastic, the stage - '50s Bauhaus. Excellent.

Sunday: Spent the morning at Ikea buying wall mounts for the kitchen. Drove back home, drilled holes in the wall and made the kitchen a little less cluttered. Quantify data from Saturday's microscopy session, clean out closet ... holly crap there is a lot of junk in there ... too much work, give-up on going to colleague's Superbowl party.

Monday: Obtain recombinant protein from a collaborator for famous last experiment. Switch buffers on the protein by diluting the sample in injection buffer and then centrifuging the sample through a 10kD centricon filter. Centrifuge is broken, run to other side of the floor and use nasty old bucket centrifuge. Spin. Dilute again. Spin. Dilute again. Spin. Perform BCA to quantify sample - spec not working ... Oh well ... Skip 1:00PM seminar. The second injection marker arrives just in time. I inject the protein (+first marker) into the cells then one hour latter reinject the same cells with RNA (+second marker) ... the needles aren't that good and the experiment is very painful. On the radio I hear about a guy at MIT who didn't get tenure and is going on a hunger strike, he's black and accusing MIT of being racist, I think about looking into it and blogging about it, but the thought quickly vanishes. At night I get home, and finish the quantitation from last week's experiment.

Tuesday: Get to lab early, wash the samples that I injected Monday and stain for injection marker #2. After labmeeting I image the famous experiment. Lots of cells. Lots of doubly injected cells. Result: no effect, not even my positive control works. Argghhh. Maybe the protein is not concentrated enough? Go to departmental "pizza talk" and fall asleep half way through ... too bad the talk was on plane of cell polarity signalling and endocytosis. Grab some coffee. Perform BCA assay on my sample. No protein? Run a gel of the sample (I kept an aliquot of the original sample from my collaborator). Frustrated I decide to abandon bench work and work on figures. I slog through Fig 1, 2, 3. I need some old data for 3, the DVD is unreadable. I head down to the Van Vactor lab (where all my 2 year old data is stored) only to find that the computer (and microscope attached to it) are occupied. I come back up, stain the gel with coomassie. It turns out that I lost all my protein during the centrifugation/concentration step. Question structure guys in the lab ... "your protein probably sticks to the filter". 7:00PM I finally get on the computer in the Van Vactor lab, rescue old data files, and then head home and get sick in the process. I work untill 11:00PM on figure 3 then head to bed.

Wednesday: I wake up at 4:30, congested, my thoughts are on what to do next. Damn protein. Do I dialyze the 50 microlitres I have left? Do I try to make the sample from scratch? Do I inject the undialyzed sample into my cells? Do I change the filament in the needle pulling machine? I pop in a cold-ease, turn on the lamp and read Paris 1919 untill 5:30. I fall back asleep. Alarm goes off at 7:00 (my wife commutes to Lowell). My wife gets up showers and makes me breakfast (espresso + bread that I baked over the weekend with ricotta and olive tapernade spread). I decide not to freak-out, and here I am writing on my blog.