My lab partner, Allison, and I have been assured that troubleshooting is a desired skill and that we will be grateful for our current Proteins woes later in our careers. And I'm proud to say that despite repeated setbacks, we've put our big-girl lab coats on, looked that ?-galactosidase in its ornery eye, and paraphrased the words of the incomparable "Rowdy" Roddy Piper in the best B movie ever, They Live:
"We have come here to chew bubblegum and purify proteins... and we're all outta bubblegum."
A run-down of the problems we've encountered and the ferreted out causes:
The exact same absorbance reading after the ?-gal assay for every single dilution of our protein. The cause, discovered after repeating the assay about a gazillion times? Our ONPG was supposed to be 4mg/mL and somehow we fouled up the concentration when we concocted the ONPG. Those puppies were burning through our substrate in seconds flat due to the low concentration.
Meandering, nonsensical Bradford assay measurements after each step of the purification process. So evidently, waaaay back in the first week or two of class, I must have had too much coffee and an unsteady hand or something, because the standard curve I'd generated using BSA dilutions as standards looked good on paper (an r2 value of 0.99 seems like it would be awesome, right?) was AGAIN off in concentration. It was just consistently off, leading to that deceptively sweet-looking curve.
A Lucy and the liquored-up bon-bon factory style mix up with data, due to us performing the assays after every purification while simultaneously repeating the assays from previous steps with our saved aliquots, trying to find our error. We found this error while figuring out the yield and seeing values increase after every step: at one point I
had a percent yield of 246%, because I am just so good at this that I can violate the principle of mass conservation and produce matter that didn't previously exist, right out of thin air.
So now here we were, feeling pretty good. It was a long road, but hey, veni, vidi, vici, right? We were all caught up and eagerly looked forward to using ion-exchange chromatography for the next round of purification. We spent all of last semester in Chromatography class, so we were totally feeling like "hey, I got this."
And then two peaks eluted off of the column. WHY IN THE NAME OF ALL THAT IS SWEET AND GOOD AND PURE IN THIS WORLD DID TWO PEAKS ELUTE OFF OF THAT COLUMN!?!?!?
With your shield, or on it, Mandy and Allison. With your shield, or on it.