User:Brigette D. Black/Notebook/Brigettes Notebook/2009/08/27/Motility Assay!

From OpenWetWare
Jump to: navigation, search

Since the NanoDrop was busted, I thought I should try to give Andy a hand in the lab. So we started out by making all of the PEM buffers, formerly known as the BRB80 series. I had already mixed up a whole bunch of BRB80-kC (0.2 mg/ml kappa casein)and had some BRB80-CS0.5 (0.5 mg/ml kappa casein). Translated, I had 1 mL aliquots of PEM-kC (at C = 0.2 mg/mL, not 0.5), so all that we needed to do was add taxol and Mg-ATP to make it a go!

With all of the buffers made, we decided to run a motility assay to check the effectiveness of the newly reconstituted rhodamin tubulin and buffers. We used the antifade I made during the summer and the new buffers. We initially mixed it up and looked at it, and unfortunately there was no movement, the antifade appeared to be not working, and some of the microtubules had buckled. So, as it turns out, I had forgotten to add dextrose to the motility solution.

Linh and I the added the forgotten dextrose to the solution and tried again. Luckily, it worked! Very nicely actually. The antifade held up for several minutes (it held up until Linh and I got bored looking at the same spot, so well over 3 minutes). The mincrotubules were really long and seemed to be pretty healthy (not a lot of breaking from what I saw). The camera was not set up on the microscope, and neither Linh nor I felt really comfortable setting it up alone (we weren't entirely sure how to, or what we could ruin on the optical tweezers). So we have no real evidence that it worked, just Larry.

I did find one microtubule that entered the field of view near the middle, and I timed that it took about 100 seconds to move across the entire field of view. The path was of course not entirely straight, but it was pretty close. But of course, this means absolutely nothing since we have no real idea what the field of view is. However, they did seem to be moving pretty quickly and holding together, so this is a good sign!