In vitro Microtubule Bundling Assay under Physiological Conditions
生理条件下的体外微管成束分析
发布: 2017年04月05日第7卷第7期 DOI: 10.21769/BioProtoc.2217 浏览次数: 8896
评审: Jyotiska ChaudhuriRebecca Van AckerElizabeth Libby
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参见作者原研究论文
The authors used this protocol in:
Apr 2016
Abstract
Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions.
Keywords: in vitro bundling assay (体外成束测定)
Fluorescence microtubules (荧光微管)
Kinesin motors (动力蛋白马达)
DEAE-Dextran coated glass chamber (DEAE-葡聚糖涂层玻璃室)
Physiological [ATP] (生理[ATP])
Background
Microtubule-based motor proteins are important as they use the chemical energy from ATP to generate force to translocate microtubules (MTs) vectorially. Of these MT based motor proteins, the superfamily known as kinesins, are responsible for directional transport and movement along microtubules. Some kinesins have MT-binding sites at both ends of the holoenzyme, so they can crosslink MTs into bundles under physiological ATP conditions (Tao et al., 2006). Due to this bundling activity, they have important roles in organizing and maintaining the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (van den Wildenberg et al., 2008). Previous bundling assays didn’t include ATP, or used a non-hydrolysable ATP analog, which could generate artificial results. Here we developed a method using physiological ATP conditions. By purifying these full-length motor proteins, it has allowed us to determine their crosslinking activity under physiological conditions.
Materials and Reagents
- Microcentrifuge tubes (1.5 ml) (Sigma-Aldrich, catalog number: Z336769 )
- Aluminum foil
- Double sided tape
- Coverslip, No. 1.5-22 x 22 mm (Sigma-Aldrich, catalog number: Z692263 )
- Microscope slides (AmScope, catalog number: BS-50P )
- Kimwipe (Sigma-Aldrich, catalog number: Z188956 )
- Pipette tips (Corning, catalog number: 4860 )
- Tubulin (CYTOSKELETON, catalog number: MT001-A )
- Glycerol (Sigma-Aldrich, catalog number: G2025 )
- Rhodamine tubulin (CYTOSKELETON, catalog number: TL590M-A )
- GTP (Sigma-Aldrich, catalog number: G8877 )
- Hydrochloric acid (HCl [37%, v/v]) (Sigma-Aldrich, catalog number: 320331 )
- Full-length kinesin motor proteins are expressed and purified from baculoviral expression system
- ATP (Sigma-Aldrich, catalog number: A6419 )
- Piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
- EGTA (Sigma-Aldrich, catalog number: 03777 )
- Potassium hydroxide (KOH)
- Paclitaxel (Sigma-Aldrich, catalog number: T7402 )
- DMSO (Sigma-Aldrich, catalog number: D2650 )
- DEAE-dextran (Sigma-Aldrich, catalog number: 30461 )
- Tris base (Sigma-Aldrich, catalog number: T1503 )
- Potassium chloride (KCl) (Sigma-Aldrich: P5405 )
- Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- Protease inhibitors
Aprotinin (Sigma-Aldrich, catalog number: A3428 )
Benzamidine (Sigma-Aldrich, catalog number: B6505 or 12072 )
Note: The product “ B6505 ” has been discontinued.
Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Leupeptin (Sigma-Aldrich, catalog number: L2884 )
Soy Bean Trypsin inhibitor (SBTI) (Sigma-Aldrich, catalog number: T6522 )
tert-Amyl methyl ether (TAME) (Sigma-Aldrich, catalog number: 283096 ) - Catalase (Sigma-Aldrich, catalog number: C1345 )
- Glucose oxidase (Sigma-Aldrich, catalog number: C6766 or G7141 )
Note: The product “ C6766 ” has been discontinued. - Glucose (Sigma-Aldrich, catalog number: G7021 or D9434 )
- 10x phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5493 )
- BRB80 (see Recipes)
- 1 µM, 10 µM, and 100 µM taxol (see Recipes)
- DEAE-dextran (see Recipes)
- Buffer T (see Recipes)
- Antifade (see Recipes)
Equipment
- Nichipet EX-Plus II pipette 2-20 μl (Nichiryo, catalog number: 00-NPLO2-20 )
- Nichipet EX-Plus II pipette 10-100 μl (Nichiryo, catalog number: 00-NPLO2-100 )
- Water bath, from ambient to 100 °C (Thermo Fisher Scientific, Thermo ScientificTM, model: PrecisionTM General Purpose Baths , catalog number: TSGP02)
- Flow chamber (see Procedure)
- Inverted fluorescence microscope (12-volt 100-watt tungsten-halide lamp, excitation wavelength, 547 nm; emission wavelength, 576 nm, in conjunction with a 570 nm dichroic mirror, objective: CFI Plan Fluor 100x oil) (Nikon Instruments, model: Eclipse E600 )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Geisterfer, Z., Tezuka, G. and Tao, L. (2017). In vitro Microtubule Bundling Assay under Physiological Conditions. Bio-protocol 7(7): e2217. DOI: 10.21769/BioProtoc.2217.
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分类
细胞生物学 > 细胞成像 > 荧光
生物化学 > 蛋白质 > 荧光
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