To make a long story short, the assay detects hydrolysis by looking at the amount of free phosphate. My PI had the idea that the protein may be more reactive if dialyzed into a different buffer solution, and discussed this with the Assistant PI, who is apparently the protein-master of the lab. Ultimately, they decided to do the overnight dialysis into PBS… otherwise known as phosphate buffered saline. That’s right, phosphate. This didn’t dawn on my PI until the colorimetric detection agent was added, and instead of a barely-detectible-by-human-eye color change, the solution in the well-plate turned instantly dark green. After a few moments of looking confused, suddenly it hit – in his case, with a response of “Oh, DUH! What were we thinking?!” Yes, the laboratory lesson of the week was, in the event of attempting to detect hydrolysis of ATP by measuring the release of phosphate, it just might not be a good idea to use a phosphate buffer! Luckily, this problem was easy to fix within 24 hours by dialyzing other fractions in a non-phosphate containing buffer, but it still provided a bit of humor throughout the lab for the rest of the week.
In other news, it has been a weekend of studying, as the biomedical graduate students are facing our first exam of the semester tomorrow. I’ve been remaining so hopeful for a snow day tomorrow which would push the exam back to Wednesday, giving me two whole extra days to study and therefore somewhat of a chance to pass. We did get about 8” of snow and knowing how inept D.C. is at dealing with snow, I thought for sure we would at least get one day off, although things seem to be clearing up well, particularly for a city which goes into panic-mode with just a few flurries. The majority of school districts in the area have already closed for tomorrow, or at least declared a two hour delay, so I’m still keeping my fingers crossed for two hours… that’s all I need! Just a two hour delay – I’m willing to go into lab and class for the rest of the day… is it really too much to ask for just two hours off? Or even a declaration of “any exams cruelly scheduled for
Silly Julie, there is no such thing as a snow day when you are a grad student :)
ReplyDeleteI hope your exam went well.