User:Andy Maloney/Water isotope effects on kinesin and microtubules - Revision history

Steven J. Koch: /* H-PEM recipe */

2011-08-27T04:15:03Z

H-PEM recipe

←Older revision		Revision as of 04:15, 27 August 2011
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	* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=151882\|ALDRICH&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC D<sub>2</sub>O]		* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=151882\|ALDRICH&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC D<sub>2</sub>O]

-	I used NaOH to pH the solution but, pH is a concentration measurement of hydrogen ions in solution and not deuterium ions. In order to measure the correct ''pD'' of this solution using the pH meter, I had to add 0.41 to the measured value (Covington 1968). This meant that the solution of PEM in D<sub>2</sub>O was pH-ed to 7.30 using NaOH. H-PEM was passed through a 0.2 μm syringe filter, aliquoted, and then stored at 4°C in screw top vials. ~~[[[~~[[User:Steven J. Koch\|Steve Koch]] 00:14, 27 August 2011 (EDT): NOTE!!! I am thinking that this is a mistake and the buffer should have been made to 6.48 !!! See: http://friendfeed.com/anthonysalvagno/dbc577ae/preliminary-tobacco-seed-growth-results-those]	+	I used NaOH to pH the solution but, pH is a concentration measurement of hydrogen ions in solution and not deuterium ions. In order to measure the correct ''pD'' of this solution using the pH meter, I had to add 0.41 to the measured value (Covington 1968). This meant that the solution of PEM in D<sub>2</sub>O was pH-ed to 7.30 using NaOH. H-PEM was passed through a 0.2 μm syringe filter, aliquoted, and then stored at 4°C in screw top vials.
		+	*[[User:Steven J. Koch\|Steve Koch]] 00:14, 27 August 2011 (EDT): NOTE!!! I am thinking that this is a mistake and the buffer should have been made to 6.48 !!! See: http://friendfeed.com/anthonysalvagno/dbc577ae/preliminary-tobacco-seed-growth-results-those
	<br style="clear:both;"/>		<br style="clear:both;"/>

Steven J. Koch: /* H-PEM recipe */

2011-08-27T04:14:27Z

H-PEM recipe

←Older revision		Revision as of 04:14, 27 August 2011
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	* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=151882\|ALDRICH&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC D<sub>2</sub>O]		* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=151882\|ALDRICH&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC D<sub>2</sub>O]

-	I used NaOH to pH the solution but, pH is a concentration measurement of hydrogen ions in solution and not deuterium ions. In order to measure the correct ''pD'' of this solution using the pH meter, I had to add 0.41 to the measured value (Covington 1968). This meant that the solution of PEM in D<sub>2</sub>O was pH-ed to 7.30 using NaOH. H-PEM was passed through a 0.2 μm syringe filter, aliquoted, and then stored at 4°C in screw top vials.	+	I used NaOH to pH the solution but, pH is a concentration measurement of hydrogen ions in solution and not deuterium ions. In order to measure the correct ''pD'' of this solution using the pH meter, I had to add 0.41 to the measured value (Covington 1968). This meant that the solution of PEM in D<sub>2</sub>O was pH-ed to 7.30 using NaOH. H-PEM was passed through a 0.2 μm syringe filter, aliquoted, and then stored at 4°C in screw top vials. [[[[[User:Steven J. Koch\|Steve Koch]] 00:14, 27 August 2011 (EDT): NOTE!!! I am thinking that this is a mistake and the buffer should have been made to 6.48 !!! See: http://friendfeed.com/anthonysalvagno/dbc577ae/preliminary-tobacco-seed-growth-results-those]
	<br style="clear:both;"/>		<br style="clear:both;"/>
		+
	====H<sub>2</sub><sup>18</sup>O overview====		====H<sub>2</sub><sup>18</sup>O overview====
	Heavy oxygen water has the following properties (Hoefs 1987, Soper 2008, Kudish 1972).		Heavy oxygen water has the following properties (Hoefs 1987, Soper 2008, Kudish 1972).

Andy Maloney: /* Purpose */

2011-06-11T20:39:01Z

Purpose

←Older revision		Revision as of 20:39, 11 June 2011
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	* [[User_talk:Andy_Maloney\|Click here to post general comments about the open dissertation]].		* [[User_talk:Andy_Maloney\|Click here to post general comments about the open dissertation]].
	* [[User_talk:Andy_Maloney/Water_isotope_effects_on_kinesin_and_microtubules\|Click here to post comments to this chapter's talk page]].		* [[User_talk:Andy_Maloney/Water_isotope_effects_on_kinesin_and_microtubules\|Click here to post comments to this chapter's talk page]].
-	~~<br style="clear:both;"/>~~

	A pdf version of the Introduction can be [http://www.openwetware.org/wiki/Image:AMDissertation_Chapter_4.pdf downloaded here]. This is the final snapshot of the dissertation that has been approved by my committee. This file will inevitably become out of sync with the wiki pages.		A pdf version of the Introduction can be [http://www.openwetware.org/wiki/Image:AMDissertation_Chapter_4.pdf downloaded here]. This is the final snapshot of the dissertation that has been approved by my committee. This file will inevitably become out of sync with the wiki pages.
		+
		+	A zip folder containing the LaTeX code can be down loaded [http://www.openwetware.org/images/9/95/Chapter_4_-_Water_istotopes.zip here].
		+	<br style="clear:both;"/>

	==Acknowledgements==		==Acknowledgements==

Andy Maloney: /* Purpose */

2011-04-12T18:27:28Z

Purpose

←Older revision		Revision as of 18:27, 12 April 2011
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	* [[User_talk:Andy_Maloney/Water_isotope_effects_on_kinesin_and_microtubules\|Click here to post comments to this chapter's talk page]].		* [[User_talk:Andy_Maloney/Water_isotope_effects_on_kinesin_and_microtubules\|Click here to post comments to this chapter's talk page]].
	<br style="clear:both;"/>		<br style="clear:both;"/>
		+
		+	A pdf version of the Introduction can be [http://www.openwetware.org/wiki/Image:AMDissertation_Chapter_4.pdf downloaded here]. This is the final snapshot of the dissertation that has been approved by my committee. This file will inevitably become out of sync with the wiki pages.
		+
	==Acknowledgements==		==Acknowledgements==
	I would like to thank Dr. Haiqing Liu (while in the lab of [http://cint.lanl.gov/gabriel_montano.shtml Dr. Gabriel A. Montano]) for supplying kinesin to our lab. I would also like to thank [http://panda.unm.edu/pandaweb/people/person.phtml?personID=76 Dr. Susan Atlas] and the support from DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008 and the UNM IGERT on Integrating Nanotechnology with Cell Biology and Neuroscience NSF Grant DGE-0549500. Finally, I'd like to thank [http://www.biotec.tu-dresden.de/research/schaeffer/ Dr. Erik Schaeffer] for his discussions on temperature stabilization.		I would like to thank Dr. Haiqing Liu (while in the lab of [http://cint.lanl.gov/gabriel_montano.shtml Dr. Gabriel A. Montano]) for supplying kinesin to our lab. I would also like to thank [http://panda.unm.edu/pandaweb/people/person.phtml?personID=76 Dr. Susan Atlas] and the support from DTRA CB Basic Research Program under Grant No. HDTRA1-09-1-008 and the UNM IGERT on Integrating Nanotechnology with Cell Biology and Neuroscience NSF Grant DGE-0549500. Finally, I'd like to thank [http://www.biotec.tu-dresden.de/research/schaeffer/ Dr. Erik Schaeffer] for his discussions on temperature stabilization.

Andy Maloney at 18:23, 12 April 2011

2011-04-12T18:23:31Z

←Older revision		Revision as of 18:23, 12 April 2011
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	\| align="left" \| [[Image:TobaccoSeeds.jpg\|300px]]		\| align="left" \| [[Image:TobaccoSeeds.jpg\|300px]]
	\|-		\|-
-	\| align="left" valign="top" width="300" \| '''Figure 1:''' Recreation of the work done by Lewis in the 1930s. The cuvette on the left has tobacco seeds that have been soaking in 100\% D~~$_2$~~O for 19 days and the one on the right has tobacco seeds that have been soaking in deionized water for 19 days. There is no apparent growth of the seeds in the left cuvette while there is definite growth in the seeds on the right.	+	\| align="left" valign="top" width="300" \| '''Figure 1:''' Recreation of the work done by Lewis in the 1930s. The cuvette on the left has tobacco seeds that have been soaking in 100% D<sub>2</sub>O for 19 days and the one on the right has tobacco seeds that have been soaking in deionized water for 19 days. There is no apparent growth of the seeds in the left cuvette while there is definite growth in the seeds on the right.
	\|}		\|}
	D<sub>2</sub>O has some rather remarkable properties that are completely different than the properties of regular water. For instance, D<sub>2</sub>O has the following properties (Hoefs 1987, Soper 2008, Katz 1957).		D<sub>2</sub>O has some rather remarkable properties that are completely different than the properties of regular water. For instance, D<sub>2</sub>O has the following properties (Hoefs 1987, Soper 2008, Katz 1957).
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	<br style="clear:both;"/>		<br style="clear:both;"/>
	====H-PEM recipe====		====H-PEM recipe====
-	No studies have been made on how water isotopes affect kinesin and microtubules. To remedy this, I took data looking at the speed at which microtubules glide at in the presence of D~~$_{~~2}$O. I prepared a solution of PEM in D<sub>2</sub>O which I called H-PEM to indicate that it contained the heavy hydrogen isotope of water. H-PEM was made in a 10x concentrated solution and contained the following.	+	No studies have been made on how water isotopes affect kinesin and microtubules. To remedy this, I took data looking at the speed at which microtubules glide at in the presence of D<sub>2</sub>O. I prepared a solution of PEM in D<sub>2</sub>O which I called H-PEM to indicate that it contained the heavy hydrogen isotope of water. H-PEM was made in a 10x concentrated solution and contained the following.
	* 800 mM of [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=80635\|SIGMA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC PIPES] (6.0474 g PIPES)		* 800 mM of [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=80635\|SIGMA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC PIPES] (6.0474 g PIPES)
	* 10 mM of [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=03778\|FLUKA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC EGTA] (.0951 g EGTA)		* 10 mM of [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=03778\|FLUKA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC EGTA] (.0951 g EGTA)
	* 10 mM of [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=M1028\|SIGMA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC MgCl<sub>2</sub>] (250 μL MgCl<sub>2</sub>)		* 10 mM of [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=M1028\|SIGMA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC MgCl<sub>2</sub>] (250 μL MgCl<sub>2</sub>)
-	* ≈ 1.25 M [http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail='prod'&productId=769764&catalogId=29104&matchedCatNo=S3185\|\|S31850\|\|S3183\|\|S3181\|\|S318500\|\|S31810\|\|S318100&pos=1&catCode=RE_SC&endecaSearchQuery=\%23store\%3DScientific\%23N\%3D0\%23rpp\%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=s318&highlightProductsItemsFlag=Y NaOH]	+	* ≈ 1.25 M [http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail='prod'&productId=769764&catalogId=29104&matchedCatNo=S3185\|\|S31850\|\|S3183\|\|S3181\|\|S318500\|\|S31810\|\|S318100&pos=1&catCode=RE_SC&endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=s318&highlightProductsItemsFlag=Y NaOH]
	* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=151882\|ALDRICH&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC D<sub>2</sub>O]		* [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=151882\|ALDRICH&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC D<sub>2</sub>O]

Andy Maloney: /* Future work */

2011-04-12T18:21:09Z

Future work

←Older revision		Revision as of 18:21, 12 April 2011
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	==Future work==		==Future work==
-	In order to measure osmotic pressure changes I can use two different osmolytes in the gliding motility assay that have very different viscosities. For example, glucose has a high viscosity as compared to betaine. Betaine is an osmolyte that your cells use to change their osmotic pressures. In order for betaine to have the same viscosity as a solution of glucose, I would have to use a lot more betaine than glucose. The concentration difference of glucose and betaine in the gliding motility assay would ~~change~~ the osmotic ~~pressure of the system~~. It will be interesting to see if I can measure speed differences using two solutions with different osmolyte concentrations that have the same viscosity.	+	In order to measure osmotic pressure changes I can use two different osmolytes in the gliding motility assay that have very different viscosities. For example, glucose has a high viscosity as compared to betaine. Betaine is an osmolyte that your cells use to change their osmotic pressures. In order for betaine to have the same viscosity as a solution of glucose, I would have to use a lot more betaine than glucose. The concentration difference of glucose and betaine in the gliding motility assay would mean that the two solutions would have different osmotic pressures. It will be interesting to see if I can measure speed differences using two solutions with different osmolyte concentrations that have the same viscosity.

-	In order to ascertain if the speed differences measured using the resealable flow cell after washing with H-PEM is due to D<sub>2</sub>O contamination, I could redo the experiment except with a lot more fluid exchange than just two sample volumes.	+	Finally, in order to ascertain if the speed differences measured using the resealable flow cell after washing with H-PEM is due to D<sub>2</sub>O contamination, I could redo the experiment except with a lot more fluid exchange than just two sample volumes.

	<font size=3>[http://www.openwetware.org/wiki/User:Andy_Maloney#Dissertation '''Return to the table of contents.''']</font>		<font size=3>[http://www.openwetware.org/wiki/User:Andy_Maloney#Dissertation '''Return to the table of contents.''']</font>

Andy Maloney: /* Conclusion */

2011-04-12T18:20:48Z

Conclusion

←Older revision		Revision as of 18:20, 12 April 2011
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	==Conclusion==		==Conclusion==
-	The goal of this experiment was to investigate the kinesin and microtubule system using different isotopes of water. Our initial thought was that by changing the water, we would change the osmotic pressure of the system. The data ~~does not indicate~~ that by changing the ~~solvent,~~ we ~~were~~ able to ~~observe~~ a ~~change in osmotic pressure~~.	+	The goal of this experiment was to investigate the kinesin and microtubule system using different isotopes of water. Our initial thought was that by changing the water, we would change the osmotic pressure of the system. The data show that by changing the isotope of water used in the motility assay caused the gliding speeds of microtubules to change in a manner that we have not been able to determine a suitable description for.

-	~~I did observe that kinesin and microtubules can measure very small changes in the viscosity of a solvent.~~ I also noted that using nail polish as the sealant for flow cells does not change the speed at which microtubules glide at. Nor does using D<sub>2</sub>O damage the system.	+	I also noted that using nail polish as the sealant for flow cells does not change the speed at which microtubules glide at. Nor does using D<sub>2</sub>O damage the system.
	<br style="clear:both;"/>		<br style="clear:both;"/>
		+
	==Future work==		==Future work==
	In order to measure osmotic pressure changes I can use two different osmolytes in the gliding motility assay that have very different viscosities. For example, glucose has a high viscosity as compared to betaine. Betaine is an osmolyte that your cells use to change their osmotic pressures. In order for betaine to have the same viscosity as a solution of glucose, I would have to use a lot more betaine than glucose. The concentration difference of glucose and betaine in the gliding motility assay would change the osmotic pressure of the system. It will be interesting to see if I can measure speed differences using two solutions with different osmolyte concentrations that have the same viscosity.		In order to measure osmotic pressure changes I can use two different osmolytes in the gliding motility assay that have very different viscosities. For example, glucose has a high viscosity as compared to betaine. Betaine is an osmolyte that your cells use to change their osmotic pressures. In order for betaine to have the same viscosity as a solution of glucose, I would have to use a lot more betaine than glucose. The concentration difference of glucose and betaine in the gliding motility assay would change the osmotic pressure of the system. It will be interesting to see if I can measure speed differences using two solutions with different osmolyte concentrations that have the same viscosity.

Andy Maloney: /* Discussion */

2011-04-12T18:20:23Z

Discussion

←Older revision		Revision as of 18:20, 12 April 2011
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	===Discussion===		===Discussion===
-	The linearity of the assays run with D<sub>2</sub>O and H<sub>2</sub><sup>18</sup>O are quite remarkable. The decrease in speed for the D<sub>2</sub>O assay is about 21% and the decrease in the H<sub>2</sub><sup>18</sup> assay is about 5%. The viscosity of D<sub>2</sub>O is about 20% greater than H<sub>2</sub>O while H<sub>2</sub><sup>18</sup> is 5% greater than H<sub>2</sub>O. The speed differences measured would suggest in light of the viscosity numbers that we measured a viscous change in the solvent and not an osmotic pressure change. ~~These~~ results are quite remarkable as they indicate that gliding motility speeds can measure small viscous changes in solutions.	+	The linearity of the assays run with D<sub>2</sub>O and H<sub>2</sub><sup>18</sup>O are quite remarkable. The decrease in speed for the D<sub>2</sub>O assay is about 21% and the decrease in the H<sub>2</sub><sup>18</sup>O assay is about 5%. The viscosity of D<sub>2</sub>O is about 20% greater than H<sub>2</sub>O while H<sub>2</sub><sup>18</sup>O is 5% greater than H<sub>2</sub>O. The speed differences measured would suggest (in light of the viscosity numbers) that we measured a viscous change in the solvent and not an osmotic pressure change. If viscosity is the cause of the decrease in speed, then these results are quite remarkable as they indicate that gliding motility speeds can measure small viscous changes in solutions. More investigation needs to be done in order to fully understand what is happening because the results may not be due to a viscous effect.

-	Kotyk et. al. (Kotyk 1990) showed that ATP activity was not affected by the addition of D<sub>2</sub>O using yeast as the test subject. Our data would suggest that ATP activity of kinesin is also not affected by the addition of D<sub>2</sub>O. If ATP activity was hindered by the addition of D<sub>2</sub>O then I would have expected to see slower speeds than what I measured using D<sub>2</sub>O ~~because clearly, there is a viscous affect and anything lower than what I measured would indicate that there was some other effect occurring~~. I did not make any measurements using less than the 1 mM ATP in the motility solutions.	+	Kotyk et al. (Kotyk 1990) showed that ATP activity was not affected by the addition of D<sub>2</sub>O using yeast as the test subject. Our data would suggest that ATP activity of kinesin is also not affected by the addition of D<sub>2</sub>O. If ATP activity was hindered by the addition of D<sub>2</sub>O then I would have expected to see slower speeds than what I measured using D<sub>2</sub>O. I did not make any measurements using less than the 1 mM ATP in the motility solutions so ATP hydrolysis was never the rate limiting step.

-	The use of nail polish as the flow cell sealant has been debated in our lab before. We use it because it is convenient and it works well. I have seen and heard of others using hot wax as the sealant and or even using glycerol. The data in Figure 3 show that using nail polish as the sealant is completely acceptable since the first cluster of data ~~point~~ gave speed measurements that were similar to those done with nail polish ~~as the sealant~~.	+	The use of nail polish as the flow cell sealant has been debated in our lab before. We use it because it is convenient and it works well. I have seen and heard of others using hot wax as the sealant and or even using glycerol. The data where I used cellophane as the flow cell sealant, shown in Figure 4, show that using nail polish as the sealant is completely acceptable since the first cluster of data points gave speed measurements that were similar to those done with nail polish.

-	Figure 3 also shows that speed measurements using 90% H-PEM do not damage the system. The speed difference between the two 0% H-PEM washings is odd however. Böhm, Stracke & Unger 2000, showed that the less kinesin there is adhered to the glass for motility, the faster microtubules will glide at. Ozeki et. al.(Ozeki 2009) also showed that two layers of casein form from creating the passivation layer on the glass. Presumably, the kinesin will either embed itself into the casein layers or adhere itself to the top layer of casein. By simply washing the flow cell with a new motility solution, I should have washed away some kinesin. This simple act should have caused the measured microtubule speeds to have increased due to the fact that there is less kinesin on the surface.	+	Figure 4 also shows that speed measurements using 90% H-PEM do not damage the system. The speed difference between the two 0% H-PEM washings is odd, however, and can probably be explained by not removing all the D<sub>2</sub>O in the flow cell. I washed the flow cell with only 2 sample volumes and there may be a change in measured speeds if more sample volumes were used in the washing.
-		+
-	~~I actually observed a speed decrease due to the washing between 0% H-PEM, 90% H-PEM~~, and ~~then 0% H-PEM. The speed decrease~~ probably ~~was caused~~ by not removing all the D<sub>2</sub>O in ~~solution~~. I washed the flow cell with only 2 sample volumes and there may be a change in measured speeds if more sample volumes were used in the washing.	+
	<br style="clear:both;"/>		<br style="clear:both;"/>

Andy Maloney: /* Results */

2011-04-12T18:16:56Z

Results

Andy Maloney: /* Data analysis */

2011-04-12T18:12:33Z

Data analysis

←Older revision		Revision as of 18:12, 12 April 2011
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	===Data analysis===		===Data analysis===
-	Please see [http://www.openwetware.org/wiki/User:Andy_Maloney/Surface_passivation_effects_on_kinesin_and_microtubules~~#Data_analysis~~ Chapter 2's ~~Data~~ analysis] section for a detailed description of how the data was analyzed.	+	Please see [http://www.openwetware.org/wiki/User:Andy_Maloney/Surface_passivation_effects_on_kinesin_and_microtubules Chapter 3]'s data analysis section for a detailed description of how the data was analyzed.
	<br style="clear:both;"/>		<br style="clear:both;"/>

←Older revision		Revision as of 18:16, 12 April 2011
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	\| align="left" \| [[Image:AM D2O.jpg\|300px]]		\| align="left" \| [[Image:AM D2O.jpg\|300px]]
	\|-		\|-
-	\| align="left" valign="top" width="300" \| '''Figure 1:''' Graph showing the gliding speed dependence of microtubules when D<sub>2</sub>O was added to the motility solution.	+	\| align="left" valign="top" width="300" \| '''Figure 2:''' Graph showing the gliding speed dependence of microtubules when D<sub>2</sub>O was added to the motility solution.
	\|-		\|-
	\| align="left" \| [[Image:AM H218O.jpg\|300px]]		\| align="left" \| [[Image:AM H218O.jpg\|300px]]
	\|-		\|-
-	\| align="left" valign="top" width="300" \| '''Figure 2:''' Graph showing the gliding speed dependence of microtubules when H<sub>2</sub><sup>18</sup>O was added to the motility solution.	+	\| align="left" valign="top" width="300" \| '''Figure 3:''' Graph showing the gliding speed dependence of microtubules when H<sub>2</sub><sup>18</sup>O was added to the motility solution.
	\|-		\|-
	\| align="left" \| [[Image:AM ReflowCell.jpg\|300px]]		\| align="left" \| [[Image:AM ReflowCell.jpg\|300px]]
	\|-		\|-
-	\| align="left" valign="top" width="300" \| '''Figure 3:''' Graph showing the gliding speed dependence of microtubules using the resealable flow cell. Data was taken using 0% concentration of H-PEM then 90% concentration of H-PEM and back to a 0% concentration.	+	\| align="left" valign="top" width="300" \| '''Figure 4:''' Graph showing the gliding speed dependence of microtubules using the resealable flow cell. Data was taken using 0% concentration of H-PEM then 90% concentration of H-PEM and back to a 0% concentration.
	\|}		\|}
-	Each slide was observed for a total of 15 fields of view, or about 30 minutes for each slide. Assays were run at 10% increments of H-PEM added to the motility solution starting from 0% and ending with 90%. Each 10% increment was then run a total of 3 times. For each slide, only the last 10 fields of view were kept to compute an average speed value for that assay. The first 5 data points were removed due to the increase in temperature of the slide from room temperature (24°C) to the objective at 33.1°C~~, see [http://www.openwetware.org/wiki/User:Andy_Maloney/Surface_passivation_effects_on_kinesin_and_microtubules Figure 7 from Chapter 2]~~. Averaging those 10 data points gave a single ~~datum~~ for the assay. Each assay was then run three separate times. This gave a total of 3 average velocity data points that were the accumulation of hundreds of tracked microtubules for a single assay.	+	Each slide was observed for a total of 15 fields of view, or about 30 minutes for each slide. Assays were run at 10% increments of H-PEM added to the motility solution starting from 0% and ending with 90%. Each 10% increment was then run a total of 3 times. For each slide, only the last 10 fields of view were kept to compute an average speed value for that assay. The first 5 data points were removed due to the increase in temperature of the slide from room temperature (24°C) to the objective at 33.1°C. Averaging those 10 data points gave a single data point for the assay. Each assay was then run three separate times. This gave a total of 3 average velocity data points that were the accumulation of hundreds of tracked microtubules for a single assay.

-	Each data point in Figure 1 is the average of those 3 speed measurements and the error bars are the standard error of the mean (SEM). The concentration of D<sub>2</sub>O was calculated by first converting the percentage values of H-PEM used in the motility assay to concentration values in one mole of H<sub>2</sub>O. Next, the baseline amount of D<sub>2</sub>O in water was calculated to be 8 mM (Somlyai 1993). The 8 mM baseline was then added to the concentration of H-PEM used.	+	Each data point in Figure 2 is the average of those 3 speed measurements and the error bars are the standard error of the mean (SEM). The concentration of D<sub>2</sub>O was calculated by first converting the percentage values of H-PEM used in the motility assay to concentration values in one mole of H<sub>2</sub>O. Next, the baseline amount of D<sub>2</sub>O in water was calculated to be 8 mM (Somlyai 1993). The 8 mM baseline was then added to the concentration of H-PEM used.

-	The assay that used D-PEM as the buffer gave a speed value that was indistinguishable from the "zero" value that ~~just used~~ H-PEM as the solution, 1015 nm/s. This result was as expected noting how linear Figure 1 is~~. The D<sub>2</sub>O contamination concentration in D-PEM was calculated to be anywhere between the maximum of 1.6 mM and minimum of 0.04 mM~~.	+	The assay that used D-PEM as the buffer gave a speed value that was indistinguishable from the ''zero'' value that had 0% H-PEM as the solution, 1015 nm/s. This result was as expected noting how linear Figure 2 is.

-	Exchanging the D<sub>2</sub>O for H<sub>2</sub><sup>18</sup>O in the motility solutions also showed a remarkably linear graph, Figure 2. The concentration of H<sub>2</sub><sup>18</sup>O was calculated in a similar fashion as was the D<sub>2</sub>O concentration.	+	Exchanging the D<sub>2</sub>O for H<sub>2</sub><sup>18</sup>O in the motility solutions also showed a remarkably linear graph, Figure 3. The concentration of H<sub>2</sub><sup>18</sup>O was calculated in a similar fashion as was the D<sub>2</sub>O concentration.

-	Data taken with the resealable flow cells that used cellophane as the "sealant" is shown in Figure 3. The small grey markers are the speed measurements for the individual assays that were conducted. The blue lines are the average speed measurements determined from those assays using the same methodology described above. The first cluster of data are speed measurements using no H-PEM in solution. The second, is when the 0% H-PEM solution in the flow cell was exchanged with 90% H-PEM. The third cluster is when the 90% H-PEM was exchanged for 0% H-PEM.	+	Data taken with the resealable flow cells that used cellophane as the ''sealant'' is shown in Figure 4. The small grey markers are the speed measurements for the individual assays that were conducted. The blue lines are the average speed measurements determined from those assays using the same methodology described above. The first cluster of data are speed measurements using no H-PEM in solution. The second, is when the 0% H-PEM solution in the flow cell was exchanged with 90% H-PEM. The third cluster is when the 90% H-PEM was exchanged for 0% H-PEM.
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		+
	===Discussion===		===Discussion===
	The linearity of the assays run with D<sub>2</sub>O and H<sub>2</sub><sup>18</sup>O are quite remarkable. The decrease in speed for the D<sub>2</sub>O assay is about 21% and the decrease in the H<sub>2</sub><sup>18</sup> assay is about 5%. The viscosity of D<sub>2</sub>O is about 20% greater than H<sub>2</sub>O while H<sub>2</sub><sup>18</sup> is 5% greater than H<sub>2</sub>O. The speed differences measured would suggest in light of the viscosity numbers that we measured a viscous change in the solvent and not an osmotic pressure change. These results are quite remarkable as they indicate that gliding motility speeds can measure small viscous changes in solutions.		The linearity of the assays run with D<sub>2</sub>O and H<sub>2</sub><sup>18</sup>O are quite remarkable. The decrease in speed for the D<sub>2</sub>O assay is about 21% and the decrease in the H<sub>2</sub><sup>18</sup> assay is about 5%. The viscosity of D<sub>2</sub>O is about 20% greater than H<sub>2</sub>O while H<sub>2</sub><sup>18</sup> is 5% greater than H<sub>2</sub>O. The speed differences measured would suggest in light of the viscosity numbers that we measured a viscous change in the solvent and not an osmotic pressure change. These results are quite remarkable as they indicate that gliding motility speeds can measure small viscous changes in solutions.