Tip 1: Myosin dead head removal techniques
Tip 2: Measuring the ATPase activity of kinesin motor proteins
For more specific technical tips please view our product pages and datasheets.
Tip 1: Myosin dead head removal techniques
To improve myosin and heavy meromyosin motility speed, try the following dead head removal techniques. Please note that Cytoskeleton, Inc. does not recommend any of the myosin or heavy meromyosin proteins for use in motility assays.
1. Mix the motor protein with 10-20 um long actin filaments and ATP at 100 mM total ionic strength, centrifuge at 100,000 x g for 1h and use the supernatant for the next step.
2. Prepare 1um long actin filaments as a blocking agent for myosin on a glass surface. Block with short actin filaments in presence of ATP. Then come back with 5 to 10 um long actin filaments (rhodamine-labeled) for motility.
The one additional trick is to add an "actin-coat" to the motility flow cell before performing the above steps. To do this, vortex some unlabeled F-actin (1uM), then give it a rinse with 1 mM ATP. This will remove some more "dead-heads" before adding the labeled actin.
This information was gathered from the following paper:
Malmqvist et al., 2004. Cardiac myosin isoforms from different species have unique enzymatic and mechanical properties. Biochemistry. 43, 15058-15065.
Tip 2: Measuring the ATPase activity of kinesin motor proteins
A straight-forward assay to examine functional changes in kinesin motor proteins is to measure the ATPase activity of kinesins by quantifying inorganic phosphate (Pi) liberation under various treatment conditions. Cytoskeleton, Inc. offers an endpoint (cat# BK053) and a kinetic(cat# BK060) assay to perform these measurements. Below are technical tips for each assay.
Kinesin ATPase Endpoint Biochem Kit (cat# BK053)
Reagents and Reaction Conditions
1) This kit contains sufficient microtubules (MTs), reaction buffers and Pi detection reagent (CytoPhos) to carry out approximately 960 standard reactions (100 μl volume). The kinesin heavy chain motor domain protein included in this kit as a control is sufficient for approximately
240 reactions.
2) Many of the reagents in this kit require reconstitution and aliquoting into convenient experiment-sized amounts. It is important to carry out the aliquoting step as freeze/thawing of some reagents (for example, kinesin protein and microtubules) is not recommended as it will result in inactivation of the reagents.
3) ATP is not included in this kit as ATP is not compatible with storage at 4°C for this protocol. The ATP product (Cat. # BSA04) from Cytoskeleton IS NOT SUITABLE for this assay. ATP can be purchased from several vendors including Sigma (Cat. # A3377). The ATP should be made to 100 mM pH 7.0 in a non-phosphate containing buffer such as 100 mM PIPES pH 7.0 and snap-frozen in 20 μl volumes. The quality of the ATP is critical for low background signal in this assay so AVOID REPEATED FREEZE/THAWS of this reagent. ATP powder should be stored at –20°C.
4) This assay is not compatible with phosphate containing buffers. If your kinesin protein is in a phosphate buffer you must remove this by dialysis or by preparing the protein in an alternative buffer such as Tris.
5) The reaction is sensitive over a range of 2 μM – 15 μM Pi (equivalent to 0.2 nmoles – 1.5 nmoles Pi in 100 μl reaction volume) and can be performed over a pH range of 6.5 – 8.5.
6) Temperature is very important for this assay. Microtubules (MTs) will depolymerize even in taxol if placed on ice. Take note when it is indicated to place a solution at room temperature.
Assay Optimization
The Kinesin ATPase endpoint assay kit has been developed to provide a good general substrate for a broad range of kinesin proteins. The control protein, kinesin heavy chain motor domain, should give a rate of 1200 – 1500 ATPs hydrolyzed per minute per mg of protein under the reaction conditions described in the manual. These values are comparable to published data. It should be noted, however, that optimization of the endpoint assay may be needed for any given kinesin protein.
There are several parameters that may particularly affect a kinesin's MT-activated ATPase activity, including:
• Protein concentration. A titration of the kinesin of interest should be performed to achieve optimal results. Kinesin protein titration is described in the Assay Protocol in the manual.
• Reaction buffer conditions. In particular, pH and salt concentration should be titrated for optimal activity. In general, final salt concentrations should be kept below 20 mM.
• ATP concentration. To minimize background readings, an ATP concentration of 0.3 - 0.6 mM is recommended. An ATP titration should be performed to obtain optimal results. ATP must be purchased separately and made up to 100 mM stock.
• Control Reactions. It is important to include control reactions in the assay, particularly if your kinesin of interest is in an impure state.
• If using a 96 well plate for this assay, we strongly recommend using a half-area well plate (180 μl volume) to perform the assays as this will maintain path length while allowing smaller reaction volumes to be used. The assay protocol will describe reactions of 100 μl final volume. If standard 300 μl volume wells are used, you will get approximately a 50% reduction in absolute values per 100 μl reaction.
Instrumentation
The assay is based upon a colorimetric change measured at 650 nm. You will require a spectrophotometer capable of measuring at this wavelength.
Kinesin ELIPA biochem kit (cat# BK060)
ELIPA Reagents
1) This kit contains sufficient ELIPA reagents to carry out approximately 100 reactions (300 ul volume). This corresponds to a complete microtiter plate of 300 ul well volume. More reactions can be achieved if half-area wells are used. The kit contains excess of the ELIPA reagents that are required to perform the standard Pi curve.
2) The kinesin heavy chain motor domain control protein contained in the kit provides sufficient reagent for 12 control assays. The remaining assays will require motor proteins that are provided by the end user. A wide selection of kinesin motor proteins are available from Cytoskeleton Inc. and can be purchased separately.
3) Many of the reagents in this kit require reconstitution and division into convenient experiment sized aliquots. It is important to carry out the aliquoting step as freeze/thawing of some reagents (for example, kinesin protein and MESG) is not recommended as it will result in inactivation of the reagents
ELIPA Optimization
The Kinesin ELIPA kit has been developed to provide a good general substrate for a broad range of kinesin motor domain proteins. For example, using this kit as outlined in the ELIPA Protocol (see manual) will result in a Vmax value of 7,000 - 8,000 n moles of ATPs hydrolysed per minute per mg of kinesin heavy chain motor domain protein. The same assay, using 235 nM of Eg5 motor domain protein results in a Vmax value of 1000 n moles of ATPs hydrolysed per minute per mg of Eg5 motor domain protein. These values are comparable to published data. It should be noted, however, that optimization of the ELIPA assay may be needed for any given motor protein or motor protein construct. It is recommended to begin optimization by titrating the amount of motor protein and ATP in the assay.
Instrumentation
The assay is based upon an absorption shift from 340 nm to 360 nm. It is therefore very important to use a spectrophotometer that has a narrow bandwidth in order that the wavelength for reading the assay does not encroach upon the 340 nm range. It is recommended that a monochromatic spectrophotometer such as a SpectroMax 350 (Molecular Devices) be used when possible as the bandwidth in these machines is very narrow (2-5 nm). If a filter based system is being used then it is important to make sure that the filter bandwidth is less than 10 nm.