Three Steps Forward, Two Steps Back

Fourth grade was a pretty crappy year for me. My teacher was simultaneously going through a bitter divorce when she learned that her teenage daughter was pregnant. She chose to take out her frustrations on us, yelling, screaming, and teaching us that no matter what went wrong in life, it was all our fault. Not caring enough to take any responsibility for her students, her answer for the somewhat disruptive class clown was to sit him next to me, and tell me I had to control him… and if he misbehaved, it was a bad refection on me. Practically every day, he would lean back on his two chair legs, the teacher would yell at him, and eventually take away his chair and force him to stand for the rest of class. Inevitably, he would wait until I was concentrated hard on reading, walk up behind me, yank the chair out from under me, and dump me on the floor to steal my chair. And, everyday, the teacher would tell me it was my fault for not doing a good job of controlling him, so my punishment was that I had to stand while he had to sit.

The one bright spot in the year was my gifted education. Unlike many places, like my husband’s school district, we did not have one completely separate class for gifted. Instead, those in the gifted program just left their regular classrooms for an hour per day. There were three of us from my classroom in the gifted program, and we were all having an equally miserable year. In an effort to drag out the time until returning back to our regular class, we used to walk down the hallway taking three steps forward, two steps backward, essentially progressing forward one step for every five steps taken.

Three steps forward, two steps back seems like the best description of my research right now. Remember the impossible purification I wrote about a few weeks ago? Well… it turned out to not me so impossible after all. One night, lying in bed unable to sleep, I had an idea… a hunch that I thought just might work. The next morning, I told my PI of my plan, to which he replied, “It won’t work. Don’t bother trying.” This statement came from the same individual who just a week before went on and on at my committee meeting about how I was a better biochemist now and had surpassed his knowledge of purification, so surely I could find a way to purify this protein. Rather than get (too) disgruntled and annoyed, I went with my gut and attempted it anyway, and two days later, smugly shoved a coomassie under his nose showing a dirty start lane and two perfectly purified bands in two separate fractions. Three celebratory steps forward!

After showing that Protein Y had properly refolded (the purification involved denaturing the protein and refolding it on the column) and still bound to Protein X, PI said the worst possible statement. “Next,” he said excitedly, “all you have to do is crosslink the lysines and that will be easy!” You see, it’s like sports. The second an announcer makes some comment like “[Quarterback] has not thrown an interception in 612 passes!” it automatically, no-fail, means that the next pass is going to be intercepted. Always. And this rule applies to PIs as well. The second he opened his mouth and described this step as “easy”, I knew I had it coming.

As it so turns out, the family of crosslinkers that will connect these proteins in the way that we need them to be connected works through linking the primary amines in the side chain of lysines. What this means is that you cannot have any amines in the buffers, because it will wind up crosslinking the buffer component rather than the proteins. And do you know what imidazole contains? Secondary amines. Harumph. So, I called up tech support at Pierce and the conversation went a little something like this.

Disgruntled Julie: Do secondary amines affect the crosslinking?
Tech Support: Yes, you can have tertiary amines, but not primary or secondary.
DJ: Is there anything I can do to get around imiazole in the buffer?
TS: Dialyze out the imidazole!
DJ: I’ve spent three years already trying to do that. Not going to happen.
TS: Have you tried doing it in steps?
DJ: 4 step, 6 step, and 8 step. Protein precipitates.
TS: How about overnight?
DJ: Precipitation.
TS: With glycerol?
DJ: With and without.
TS: How much glycerol?
DJ: 10%, 20%, 30%
TS: Spin column?
DJ: Precipitates.
TS: Desalting column?
DJ: Precipitates.
TS: Well, shit.

Indeed. My sentiments exactly. I finally have both proteins purified, and I cannot crosslink them because I absolutely, positively have not been able to dialyze the imidazole out of the protein.

O n e. T w o. Two giant steps backward.