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| ==Chemicals==
| | * [http://www.proweb.org/kinesin/Methods/motility.html Hancock] |
| ====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=80635|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC PIPES]====
| | ** 80 mM PIPES |
| Somethings to note about PIPES is that it is an acid. Don't get this stuff on your skin because it will irritate it. We use acid PIPES because there was a discussion with Koch about what type of ions are needed in solution for motility to work. Some say that you need potassium and others, sodium. Since we have a free acid form of PIPES, we can add either NaOH or KOH to pH the buffer to PIPES's pK<sub>a</sub> of 6.89 thus adding the appropriate counter ion we want.
| | ** 1 mM MgCl<sub>2</sub> |
| | | ** 1 mM EGTA |
| PIPES should be stored in the desiccator at room temperature in its original container.
| | ** pH 6.85 |
| ====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=03778|FLUKA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC EGTA]====
| | * [http://cshprotocols.cshlp.org/cgi/content/full/2006/20/pdb.rec10559?text_only=true Cold Spring Harbor Labs] |
| EGTA is also an acid so be careful. The reason we use the acid form is for the same reason we use the acid form of PIPES. It should be stored in its original container in the desiccator. | | ** 100 mM PIPES |
| ====[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=M1028|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC MgCl<sub>2</sub>]====
| | ** 2 mM EGTA |
| MgCl<sub>2</sub> is very hygroscopic. This means it really likes to be in water and it will pull moisture from the atmosphere so it can be in it. For this reason, we use a 1 M solution of MgCl<sub>2</sub> in water. This way we do not have to fuss around with trying to weigh out an appropriate amount of MgCl<sub>2</sub>, since what we weigh would also include water. | | ** 1 mM MgSO<sub>4</sub> |
| | | ** pH 6.95 |
| MgCl<sub>2</sub> should be stored in its original container at room temperature. You do not have to desiccate it since the MgCl<sub>2</sub> is in solution already.
| | * [http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-application/protocols/immunofluorescence.html Sigma Aldrich] |
| ====[http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&productId=769764&catalogId=29104&matchedCatNo=S318500||S3183||S3185||S31850||S31810||S318100||S3181&pos=1&catCode=RE_SC&endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=s318&highlightProductsItemsFlag=Y NaOH]====
| | ** 100 mM PIPES |
| Be careful with this. It is a strong base. NaOH is again hygroscopic and thus likes to pull moisture from the atmosphere. I store it in its original container and in the desiccator. When ever I open the jar and close it, I blow in nitrogen so it is stored in a nitrogen rich environment (i.e. no moisture). It is a strong base so be careful when handling it. I like to make up 1 [http://en.wikipedia.org/wiki/Concentration#Normality Normal] solutions of this and use it in this form.
| | ** 5 mM EGTA |
| | | ** 2 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O |
| When making a 1 N solution, I typically store it in a glass jar. I sometimes will also make up a 12 N solution and dilute it from that but, this is not necessary.
| | ** pH 6.8 |
| | | * [http://www.bcm.edu/microscopy/?PMID=2855 Bayer College of Medicine] |
| ==Recipe==
| | ** 80 mM K-PIPES |
| * 80 mM [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=80635|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC PIPES] | | ** 5 mM EGTA |
| * 1 mM [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=03778|FLUKA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC EGTA] | | ** 2 mM MgCl<sub>2</sub> |
| * 1 mM [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=M1028|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC MgCl<sub>2</sub>] | | ** pH 6.8 |
| * ~ 125 mM [http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&productId=769764&catalogId=29104&matchedCatNo=S318500||S3183||S3185||S31850||S31810||S318100||S3181&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.ece.cmu.edu/~yuliwang/Methods/Materials/Fluorescent%20Labelilng/RhodamineTubulin.pdf Yu-li Wang from Carnegie Mellon] This is a pdf file. |
| * pH 6.89 | | ** 100 mM PIPES |
| ==Notes==
| | ** 1 mM EGTA |
| Always put PIPES and EGTA into water and not the other way around. Once you have all of your PIPES and EGTA in some water, go ahead and add around 120 mM of your 1 N [http://www.openwetware.org/wiki/User:Andy_Maloney/Kinesin_%26_Microtubule_Page/1_N_NaOH NaOH] stock solution. I had previously been doing this, however, I think I like weighing out an amount of NaOH that will be less than 125 mM concentration in my final solution and just adding the appropriate amount of the 1 N solution to get it to the ~120 mM.
| | ** 0.5 mM MgCl2 |
| | | ** pH 6.9 |
| You will see that before the NaOH is added, PIPES and EGTA will not go into solution. After adding it, it will. Keep adding small amounts of NaOH into solution until all the PIPES and EGTA is dissolved (the total amount is approximately 125 mM). If you are lucky, you will get really close to the pH that the buffer needs to be at once all the PIPES and EGTA is in solution. Add the remaining amount of water needed and pH the solution till you get it to a pH of 6.89.
| | * [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T0G-4JKHMBD-4&_user=1550512&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=937840362&_rerunOrigin=google&_acct=C000053660&_version=1&_urlVersion=0&_userid=1550512&md5=a51e4c2c3d082850d44f8d0824033a88 Tedeschi of Italy] |
| | | ** 85 mM Pipes |
| If, you make the solution too basic, just add small amounts of [http://www.fishersci.com/wps/portal/PRODUCTDETAIL?prodcutdetail=%27prod%27&productId=667950&catalogId=29104&matchedCatNo=A144500||A144C212||A144500LB||A144S500||A144612GAL||A144SI212||A144212||A144S212||A144P19&pos=1&catCode=RE_SC&endecaSearchQuery=%23store%3DScientific%23N%3D0%23rpp%3D15&fromCat=yes&keepSessionSearchOutPut=true&fromSearch=Y&searchKey=a144&highlightProductsItemsFlag=Y HCl] to get the pH right.
| | ** 10 mM EGTA |
| | ** 1 mM MgCl<sub>2</sub> |
| | ** pH 6.94 |
| | * [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B94RW-4TX1P02-8&_user=1550512&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000053660&_version=1&_urlVersion=0&_userid=1550512&md5=a4a0f0dc1f0a705362349f6ef05e3489 Steve Gross of U. Minnesota with Steve Block] |
| | ** 80 mM Pipes |
| | ** 1 mM EGTA |
| | ** 4 mM MgCl<sub>2</sub> |
