Talk:Koch Lab:Protocols/Kinesin

Water purification and Ca++ ion discussion
Andy Maloney 01:53, 17 April 2009 (EDT): Should we get a water purifier that makes 18.2 MΩ-cm H2O? That way we don't have to worry about stray ions mucking up our buffers? With it, we shouldn't have to use EGTA. Besides, if we are wanting to chelate calcium, why aren't people using BAPTA? It's affinity for calcium is huge compared to EGTA. When I was calibrating indo-1 (which has a Kd for calcium in the tens of nM range) this is what I used.

Steve Koch 02:00, 17 April 2009 (EDT): Yes we probably should. I had talked to Dan about this a couple years ago and there was some issue with plumbing. The building has 18.2 MΩ-cm water, but it's not plumbed to our lab. I'm not sure how 18.2 it is, and Carboys are annoying (but not too bad). I believe it was going to be tough to get DI water to feed into the purifying, if I remember correctly? Can you ask Dan about it? Evan is interested too. As for the EGTA, there is a whole lot of magic that goes into the kinesin assays. This magic took a whole bunch of time to develop by the previous researchers. I don't doubt that there's ways to improve it. But we should start with the magic that works and then try tweaking later when we have time. I may be wrong about the calcium and EGTA legacy. BTW: the divalent contaminants wouldn't be coming from the water, but other sources. The main source would be (in my mind), the very casein that is required to magically stabilize the motility assay. One function of casein (milk protein) is to deliver calcium. So, it wouldn't surprise me if casein has a lot of Ca++ in it. If you're interested, I have a whole bunch of casein research somewhere.

Andy Maloney 02:11, 17 April 2009 (EDT): Yes. I am interested in the casein stuff. The worst case scenario is that I can "filter" the casein by titrating it through an ion exchange column. Using a matrix like Dowex does a remarkable job of getting rid of calcium ions, even if they aren't specifically in solution if I remember correctly. Of course, this means we will need to exchange the ions with something else like Na or K. Why do we use casein? Is it used specifically for a cover for the glass? If that's the case, why don't we just silanize the glass? That way, we don't have to deal with calcium in at all. As for the nanopure water, we can get a purifier for I think less than $5k from VWR. That way we will have the purifier next to the sink, no plumbing necessary and we can produce several litters / day. Finally, I'm all for doing legacy if it makes sense. But, once I understand more about the process, I may want to upgrade legacy buffers to super awesome kick ass Koch Lab 2.0 buffers.

Andy Maloney 02:27, 17 April 2009 (EDT):
 * Water purifier. I'll see if Rick can get us a better deal.


 * Steve Koch 02:32, 17 April 2009 (EDT):We're on the same page -- get legacy going, then try for kick ass 2.0 when have a foothold (or should I say headhold). You know more about the ion stuff than I do.  Those columns may be a great idea.  One other thing about the magic casein is that Sigma discontinued the specific product a lot of people used!  It would be hella nice to understand more about the surface chemistry.  One idea for gliding assay would be to use the pentahistidine antibody and pentahistidine-tagged kinesin that Block lab used.  (antiPenta-his is strong, whereas Ni-NTA/his is very weak).
 * Steve Koch 02:32, 17 April 2009 (EDT): Don't you need to run the purifier off of a distilled water source? I.e., not tap water?
 * Andy Maloney 02:35, 17 April 2009 (EDT): Nope. I got this system for my old lab and it works quite well. Using the prefilter system prolongs the filter life spans but it makes 18.2 MΩ-cm water from tap.
 * Steve Koch 02:39, 17 April 2009 (EDT):Oh, then great! Yes we do want one.  Let's ask Evan if he has any preferences (doubt he will).  I think we should hang it above or near the new sink, though,...we can think about it.  I don't have any brand preferences, but I've only used Barnstead in the past.  It was delicious.
 * Andy Maloney 02:44, 17 April 2009 (EDT): Barnstead is the one that makes the unit I linked to above. I've already asked Rick for a quote. He usually gets back to me within a day. Should we ask Evan? I've used this system before and set it up so I know how to do it. Plus, I know it works. Oh, I forgot Evan is here. So yes, we should ask him if you want to.

Andy Maloney 13:00, 17 April 2009 (EDT): I've got the quote up on Google docs and I've filled out the paperwork. All I need is a signature.