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Submitted by Kristine Cerbone on Fri, 11/15/2013 - 3:31pm
If it’s one thing I’ve learned in my technique classes is that there is a lot of ‘hurry up and wait’ procedures. Working with proteins is definitely no exception.
For the past month or so my protein class has been working on purifying bacterial alkaline phosphatase (BAP). Rewinding back to day one of the process, we received our bacteria that had already been induced so our first step was to lyse the cells. Method used for lysing = sonication. I have to admit, this is my least favorite method for lysing cells. The sound of the sonicator can definitely make my skin crawl.
Fast forwarding past a few other preparation steps for this purification (pouring an affinity column, making buffers) we come to adding our bacterial samples to our column. After spinning the column on a rotating wheel, the real fun began. Using a denaturing binding buffer and a denaturing elution buffer collected 1ml fractions as they came off the column. This was like watching paint dry. While trying to behave ourselves while we were collecting fractions, my lab partner and I began to time how long it took to collect 1ml. On average it took 5:01mins. We collected 16 fractions. The good news is, paint will dry eventually.
Once our fractions were collected we ran them on an SDS gel, which gave us some promising results that we were on the way to purifying the correct protein. However, by this point our protein was denatured, so now is the time to refold it. Choosing the two most promising looking fractions according to the gel, we put the samples thru dialysis to refold our proteins.
The problems with Bradford
Next step: Bradford Assay. Things were running a little too smoothly, so we should have expected something to set us back a little. Somehow, wither there wasn’t enough caffeine in our systems or because it was just an ‘off’ day. We miss calculated and made a 75x dilution. I felt pretty dumb when I realized what we had done, but on the other hand it was kind of funny. So, we couldn’t really trust the results of our Bradford. We recalculated and made a 10x of our sample to redo our Bradford. However our lab woes weren’t over. Note to self, always double or triple check the settings on your equipment before use. During the 2nd attempt of our Bradford, we took our readings at the wrong wavelength. But hey, third times a charm right?
To fast forward again past another dialysis, we ran an ion exchange column. At least this column didn’t take as long as the first and after every fraction, we took a small sample and mixed in substrate to see if we had a reaction. SUCCESS! It’s always fun to see that it looks like your heading in the right direction before the anaylisis gel is ran.
Our SDS gel came out pretty too, just a little ripped.