Archive for June, 2009

Dye by Night

June 28, 2009

The Order Of Science Scouts: “Inappropriate Nocturnal Use of Lab Equipment in the Name of Alternative Science Experimentation / Communication” badge

Due to the clandestine nature of the activity, meticulous notes were not recorded by the principal investigator. Therefore, details are lacking and experimental procedures are  incomplete. This work is mainly reported because its the middle of Summer and I can’t sleep.

Background

The girl I’d had my eye on when I was an undergrad worked in a laser lab. Due to malfunctioning  equipment, a large quantity of floor wax in the lab and a portion of the hallway outside had become contaminated with rhodamine.

Experimental

Work was not undertaken until everyone in the department had gone home for the night in order to prevent goggle eyed stares from passers by demanding to know just what it was I thought I was doing.

A single edged razor and the better part of an hour were used to strip approximately 5 g of electric-pink floor wax from the hallway tiles*. It was surmised that since the custodians had long eschewed the department on the grounds that chemicals routinely leap from sealed bottles and kill people, this vandalism to the floor would not be particularly bemoaned.

The pink waxy solid was dissolved and filtered to remove traces of lint, staples, and gravel. An acid-base extraction was used to separate the rhodamine from the matrix, and the solution was concentrated in vacuo. Another hour was spent playing with chromatography conditions and purifying the product which came off the column in a fluorescent band like some sort of sophomore organic lab demo, yielding several mg of crystals which were sealed in a vial and presented to her for her birthday.

Results and Conclusions

It didn’t work out though, and for the best. She ended up dating someone who even though I tried my best to be jealous of at first, I have to admit was a great guy.

I graduated and got accepted to a program where I met a girl who I am deliriously happy with. However, when I reflect upon all the ill advised nocturnal misappropriations of scientific equipment I am guilty of perpetrating, this one stands out as not the most dangerous, but probably the silliest. Eventhough, they do say we regret most that which we do not try.

*It should be noted that in many older buildings, 9-inch floor tiles may contain asbestos. Keep this in mind before tearing them up with razor blades in order to impress a girl

Chromophores and You

June 3, 2009

Chances are if you work in a lab that does any sort of biochem related activities, at some point you’ll have to stick something to a protein. The two most popular points of attachment are primary amines and thiols because they’re fairly nucleophilic under conditions which won’t completely destroy your protein.

protein001

Primary amines are most often conveniently encountered in the form of lysine residues, or N-termini. In fact, most proteins are lousy with the damn things which is a problem if you want site specificity. The N-terminus tends to be more nucleophilic than other sites, but you’ll generally end up drenching your protein with the reactive compound and hope it doesn’t land anywhere that it will get in the way. Thiols show up as cysteine residues, and unlike primary amines tend to be much rarer and quite a few proteins have none at all naturally. If you know the site where you want something attached, it is fairly easy to engineer a cysteine there for site specificity.

thiol_labeling

Methods for modifying thiols tend to come down to two options: forming a thioether, or forming a disulfide. The most popular groups for the first are iodoacetamides, and maleamides. The third usually uses a thiosulfonyl. However, thioethers are pretty irreversible, and disulfides usually get reduced somewhere along the line. So how do you nail something onto a thiol but leave yourself the option of selectively removing it later?

488_phenylmercury

That’s phenylmercury, baby! Stuck to Alexa Fluor® 488. This autism campaigner’s wet dream of a chromophore is available from Invitrogen. That S-Hg bond will stand up to TCEP reduction, but can be knocked off by DTT or 0.1 M HCl.

Can’t say I have any plans to rush out and use this thing though. I’ve got much better means of killing brain cells.