User:Brigette D. Black/Notebook/Brigettes Notebook/2009/07/08/Variable Time with Malachite Green: Attempt 1

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I started off the day by talking with Andy about what I had been doing with the assays thus far and what my immediate plans were. It turned out to be pretty helpful in that it forced me to explain what I was doing and why, which helps clears out the muck in my head and crystalize plans. He also explained a little of the chemistry behind the assay, which is also extremely helpful to my understanding.

I went into lab and had a similar conversation with Koch. We calculated the approximate amount of steps the kinesin was taking in my samples from yesterday in the following way:

Guess-timating Kinesin Activity

Molarity of kinesin = ~5*10^4 g/mol * 5 ug/mL = 100 nM (Steve Koch 00:28, 10 July 2009 (EDT): I think you mean "/" not *)

  • Andy Maloney 23:24, 9 July 2009 (EDT): I don't understand this equation. What are you multiplying together?

Each kinesin should take one step in a cycle, each step produces 1 Pi, so in one cycle we should see about 100 nM of phosphate produced.

  • Andy Maloney 23:24, 9 July 2009 (EDT): I'm not sure what a cycle is but, a kinesin molecule hydrolyzes 1 ATP molecule per step. If indeed you have 100 nM of kinesin molecules in your sauce then you will get 100 nM of Pi if every kinesin molecule takes one step.
    • Steve Koch 00:28, 10 July 2009 (EDT): I think you guys are saying the same thing here & I agree with you. Above, Brigette is roughly saying kinesin monomer weighs 50 kilodalton (5E4 g/mol) and concentration is 5E-6 g / mL = 5E-3 g/L. So, dividing these give molarity of kinesin = 1E-7 M.

Yesterday, I measured that there was about 25 uM phosphate produced in about 30 minutes, which means that the kinesin is doing 250 cycles in 30 minutes.

In a gliding motility assay, we want to see the microtubules moving about 800 nm/s. Each step is 8nm, so for our purposes we would like to see kinesin that preforms 100 cycles/second. Obviously, our kinesin is way way off this mark.

Koch also mentioned that for this measurement we wanted more microtubules than kinesin. I looked up the weight of tubulin, about 5*10^4 g/mol, like kinesin. We have tubulin at 5 mg/mL, in 5 uL aliquots, for a total of 25 ug / aliquot. Once in motility solution, we had 25 ug/mL or 500 nM. This is about 5 times the about of kinesin in our solution, but this is calculated just with tubulin, not MTs. I am not sure of how to calculate the number MTs, other than with a BioSpin column, of which we do not have the proper size.

  • Andy Maloney 23:24, 9 July 2009 (EDT): What's this BioSpin column thing? After thinking about this some more, our calculations may be wrong. We can talk more tomorrow and you can tell me about this BioSpin prep.

In order to reconcile any potential lack of MTs, I decided to triple the concentration of them today, just to be safe.

Kinetic Assay

Koch let me know that antifade is not needed in our spectrophotometry measurements, so today I made the microtubules by adding....

  • 5uL unlabeled tubulin + 95 uL BRB80T (to stabalize) + 2uL BME + 258uL BRB80
  • Total = 350 uL (enough for about 12 reactions at 3 times concentration)

I grabbed some kinesin from the freezer, and in order to draw out a microliter, I had to dilute it a bit. I added 4 uL to the tube, thus diluting it to 1 mg/mL. I pulled out 2 uL of this, and added 400 uL of BRB80. I put the other 3 uL of now 1 mg/mL back into the -80 freezer.

I decided to use 2mM ATP, instead of 1mM, as from what I saw yesterday it seemed to give a higher activity level. I assigned the cuvettes as follows:

  • Cuvette 1: MTs + 2mM ATP +BRB80 (0 minutes)
  • Cuvette 2: MTs + ATP + Kinesin (0 minutes)
  • Cuvette 3: MTs + ATP + Kinesin (10 minutes)
  • Cuvette 4: MTs + ATP + Kinesin (20 minutes)
  • Cuvette 5: MTs + ATP + Kinesin (30 minutes)
  • Cuvette 6: MTs + ATP + Kinesin (50 minutes)
  • Cuvette 7: MTs + ATP + Kinesin ( 60 minutes)
  • Cuvette 8: MTs + ATP + Kinesin (85 minutes)
  • Cuvette 9: MTs + ATP + Kinesin (110 minutes)
  • Cuvette 10: MTs + ATP + Kinesin (Overnight, about 18 hours)
  • Cuvette 11: MTs + ATP + Kinesin (24 hours)
  • Cuvette 12: MTs + ATP +BRB80 (24 hours)

Cuvette 1 is supposed to be a blank, to see how much Pi is in kinesin, and to give an idea of what non hydrolyzed ATP absorbs. The last cuvette, 12, is meant to give an idea of to what extent ATP will hydrolyze on its own.

I made the samples by adding 30 uL MT solution, 30 uL of Kinesin or BRB80, and 1.2 uL of 100 mM ATP . I let the reactions occur in the cuvettes, and then added quencher (perchloric acid) when the reaction time had been reached.

I asked the guys in the lab if I could keep the door propped open so that I could go in and out between the labs, and since I was going to be there until about 8:30 making measurements. However, right after I quenched Cuvette 6, I discovered the door had been closed and everyone had gone home. When making the standard curve, I had left the reactions sitting in quencher overnight, and the results were terrible. So I think it is safe to say that these reactions are pretty close to dead. I will try again tomorrow... Steve Koch 00:31, 10 July 2009 (EDT): Wow, I am really sorry this happened. That is just awful to lose all that work.