Effects of Surface Passivation on Gliding Motility Assays

Maloney, Andy; Herskowitz, Lawrence J.; Koch, Steven J.

doi:10.1371/journal.pone.0019522

Cited by 39 publications

(44 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Microtubules were polymerized from commercially available porcine tubulin following a protocol described in previous work (Maloney et al, 2011). For fluorescence imaging, we used a 7:3 mixture of bare and rhodamine-labeled tubulin (Cytoskeleton, Inc.).…”

Section: Microtubule and Kinesinmentioning

confidence: 99%

“…Activity of molecular motors fixed on a surface is very sensitive to interaction with the surface. For the optimal functionality and effective consumption of the kinesin proteins in microtubule gliding assays, casein is typically employed to pretreat glass surfaces (Maloney et al, 2011). However, inclusion of casein in our experimental procedure often suppressed binding of microtubules to the kinesin-treated surface, possibly due to a non-optimal casein solution.…”

Section: Motility Assaymentioning

confidence: 99%

See 1 more Smart Citation

Microtubule shuttles on kinesin-coated glass micro-wire tracks

Kim

Liao

Sikora

et al. 2014

Biomed Microdevices

View full text Add to dashboard Cite

Gliding of microtubule filaments on surfaces coated with the motor protein kinesin has potential applications for nano-scale devices. The ability to guide the gliding direction in three dimensions allows the fabrication of tracks of arbitrary geometry in space. Here, we achieve this by using kinesin-coated glass wires of micrometer diameter range. Unlike previous methods in which the guiding tracks are fixed on flat two-dimensional surfaces, the flexibility of glass wires in shape and size facilitates building in-vitro devices that have deformable tracks.

show abstract

Section: Microtubule and Kinesinmentioning

confidence: 99%

Section: Motility Assaymentioning

confidence: 99%

Microtubule shuttles on kinesin-coated glass micro-wire tracks

Kim

Liao

Sikora

et al. 2014

Biomed Microdevices

View full text Add to dashboard Cite

show abstract

“…8 The microtubules solution is first inserted into a standard flow cell, 9 and incubated for 5 min. The cell is then flushed with BRB80 buffer, 10 containing 10¯M taxol, antifade solution, and 0.2 mg mL ¹1 casein, to remove unbound microtubules and prevent QD adherence.…”

mentioning

confidence: 99%

Quantum Dot Motion on Microtubules

et al. 2012

View full text Add to dashboard Cite

Kinesin is one of the numerous motor proteins present in the cell. Its function is to transport cargo along microtubules, which serve as tracks, by efficiently making use of chemical energy. Here, we have studied the translocation of quantum dots by kinesin. Kinesin was bacterially expressed and conjugated to quantum dots, thus allowing observation of the motion of the quantum dots along the microtubules by fluorescence microscopy. The velocity was determined by a kymograph, and was in agreement with expectations.Where there is life, there is motion. Our own movements, for example, are the result of motor proteins. One of the best studied examples, kinesin, "walks" along biopolymer tracks termed microtubules, and facilitates intracellular cargo transport (Figure 1).Kinesin is a heterotetramer constructed from two light chains and two heavy chains, which dimerize to form the stalk. They are terminated by a motor head, which binds specifically to the tubulin, and forms the constitutive element of the microtubule, providing the step-like walking motion. The biological track is a continuously evolving structure and displays polarity. It polymerizes rapidly at the "plus" end and slowly at the "minus" end. 1 The majority of kinesin motors such as kinesin-1, move toward the plus end.The mechanical energy required for the walking motion is fuelled by adenosine triphosphate (ATP) hydrolysis, where a single ATP molecule is consumed for each 8 nm step.1,2 The chemical energy is converted into mechanical energy with a high efficiency (ca. 50%).3 Furthermore, kinesin can translocate several piconewtons of load over relatively long distances, and its activity is barely limited by cargo size. 4 The velocity can be controlled by the ATP concentration.5 Overall, these properties make kinesins interesting macromolecules for lab-on-a-chip devices. In the present report, the transport of streptavidin-coated quantum dots (QDs) by kinesin is studied. The advantage of QDs is their small size (ca. 20 nm) and enhanced photostability as compared to conventional fluorophores, 6 thus allowing the direct confirmation of both the activity of the expressed kinesin and its ability to translocate cargo.Truncated and biotinylated kinesin-1 (length: 400 amino acids) was expressed in E. coli using a standard protocol, 2 with histidine tags to facilitate purification. To enable cargo attachment, the strong binding interaction between streptavidin and biotin was exploited.7 The microtubules were labeled with rhodamine to allow their identification with fluorescence microscopy. An Olympus IX-71 microscope, equipped with a Hamamatsu ImagEM CCD camera, was used for the observations. Streptavidin-coated QDs were purchased from Invitrogen. Unlabeled tubuline and rhodamine-labeled tubulin were purchased from Cytoskeleton Inc.A schematic representation of the motility assay, showing the QDs that are attached to the kinesin, is presented in Figure 2. In the presence of ATP, the motor will spontaneously attach to the microtubule and "walk" along its length. ...

show abstract

“…The tryptic digest of commercially available casein, containing all three variants in the ratio of 4:1:4:1 (αS1:αS2:β:κ) was employed (Maloney et al 2011). The digestion was carried out in the presence of 50 mM ammonium bicarbonates, Dithiothreitol (DTT) (45 mM, 50 μL) and IAA (100 mM, 50 μL), which causes high salt concentration in the sample.…”

Section: +mentioning

confidence: 99%

Development of new multifunctional terpolymer sorbent for proteomics applications

Najam‐ul‐Haq

Saeed

Jabeen

et al. 2014

Biomedical Chromatography

View full text Add to dashboard Cite

Determination of the availability of phases for specific separations is an important task achieved by a separation chemist. This becomes vital when the complex samples like biofluids are dealt with in proteome science. The work presented here involves the synthesis and application of terpolymeric sorbent with different functionalizations adopted for the selective enrichment of biomolecules of interest from biological fluids. Synthesis of terpolymer was carried out by the radical polymerization of monomers: methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross-linking agent, benzoyl peroxide as initiator and chloroform as a porogenic solvent. Characterization was done through Fourier transform infrared spectroscopy, scanning electron microscopy and nitrogen adsorption porosimetry. The polymer was further modified to immobilized metal ion affinity chromatographic material, with immobilized Fe(3+)/La(3+) ions that allowed phosphopeptide enrichment from tryptic digests of standard proteins as well as milk, egg yolk and human serum. Sensitivity of enrichment down to 50 fmol was achieved in the presence of complex protein background as bovine serum albumin. Hydrophobicity was introduced through octadecyl amine, which provides comparable results to ZipTip C18/C4 for desalting of complex mixtures of all caseins. Analysis of the enriched content was performed by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

show abstract

Effects of Surface Passivation on Gliding Motility Assays

Cited by 39 publications

References 43 publications

Microtubule shuttles on kinesin-coated glass micro-wire tracks

Microtubule shuttles on kinesin-coated glass micro-wire tracks

Quantum Dot Motion on Microtubules

Development of new multifunctional terpolymer sorbent for proteomics applications

Contact Info

Product

Resources

About