Microtubule Binding Protein Spin-Down Assay Biochem Kit

Microtubule Binding Protein Spin-Down Assay Biochem Kit
$0.00

Product Uses Include

  • To determine whether a protein or compound binds to microtubules.
  • To test various deletion mutants for their ability to still bind microtubules, then calculate their affinity for microtubules, and hence identify the microtubule binding site.
  • In the presence of a non-hydrolyzable analog of ATP (kinesins) or GTP (dynamin) extract these from cells or show their binding affinity when mutated.

Introduction
This assay allows the identification of proteins that will bind to microtubules (MTs) in vitro.  The assay relies on the fact that MTs will pellet when centrifuged at 100,000 x g. Therefore, any protein that is associated with the MTs will pellet with them during centrifugation. A simple SDS-PAGE analysis of the supernatant versus pellet fraction will identify if a protein is able to associate with MTs.  The assay description given in this manual is for recombinantly expressed “test” proteins, however, the assay can be adapted for cell lysates or in vitro translation products.

It should be noted that in vivo confirmation of MT association should be obtained in order to confirm that the protein can be classified as a MAP.  This association need not occur throughout the whole cell cycle and may even be developmentally regulated, indeed transient association of MAPs with microtubules is the norm rather than the exception.

Kit contents
The kit contains sufficient materials for 30-100 assays depending on assay volume. The following reagents are included:

  1. Tubulin, >99% pure, (Cat. # T240)
  2. Microtubule associated protein fraction (positive control) (Cat. # MAPF)
  3. Bovine serum albumin (BSA) protein (negative control)
  4. General tubulin buffer (Cat. # BST01)
  5. Tubulin glycerol buffer (Cat. # BST05)
  6. GTP (Cat. # BST06)
  7. Microtubule resuspension buffer
  8. Paclitaxel (Cat. # TXD01)
  9. DMSO
  10. Salt-extraction buffer
  11. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. TCA solution 50% (w/v) for protein precipitation if necessary.
  2. Centrifugation set-up capable of 100,000 x g at 4°C and 24°C, 50 -200 µl volume capacity.
  3. SDS-PAGE system.
  4. Detection system for protein of interest (coomassie is good for purified proteins, Western blot or silver stain for less pure or low abundance test proteins).
  5. Gel scanner for densitometric determinations.

Example results
The microtubule binding spin-down assay was tested by combining MTs with the a microtubule associated protein fraction (Cat. # MAPF) or with BSA. As expected, MAPFs co-sediment with the microtubules while BSA does not (Fig. 1)

bk029fig1

Figure 1. MAP Spin-Down Assay using BK029. Microtubules were mixed with buffer (MT), MAP proteins (MT + MAPF) or BSA (MT +BSA) and MTs were pelleted by centrifugation at 100,000 x g. Supernatant (S) and Pellet (P) fractions were examined by SDS-PAGE. MAP proteins, but not BSA, bind to MTs and co-precipitate. MAP (MAPF) or BSA (BSA) proteins alone do not pellet.

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Hori, Tetsuya et al.Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse modeleLife2022ISSN 2050--084X
Wortzel, Inbal et al.Mitotic HOOK3 phosphorylation by ERK1c drives microtubule-dependent Golgi destabilization and fragmentationiScience2021ISSN 2589-0042
Huang, Ning et al.Reprogramming an energetic AKT-PAK5 axis boosts axon energy supply and facilitates neuron survival and regeneration after injury and ischemiaCurrent Biology2021ISSN 1879-0445
Li, Yan et al.The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule bindingJournal of Biological Chemistry2021ISSN 1083-351X
Miquel, M. et al.Microtubule interaction of LICC1, a maize homologue of a component of the human muskelin/RanBPM/CTLH protein complexhttp://bp.ueb.cas.cz/doi/10.32615/bp.2020.168.html2021ISSN 0006-3134
Grubisha, M. J. et al.MAP2 is differentially phosphorylated in schizophrenia, altering its functionMolecular Psychiatry 2021 26:92021ISSN 1476--5578
Woo, Jung AA et al.β-arrestin1 promotes tauopathy by transducing GPCR signaling, disrupting microtubules and autophagyLife science alliance2021ISSN 2575--1077
Shih, Pu Yun et al.Autism-linked mutations of CTTNBP2 reduce social interaction and impair dendritic spine formation via diverse mechanismsActa neuropathologica communications2020ISSN 2051-5960
Ait-Bouziad, Nadine et al.Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- And lipid-binding properties of TauJournal of Biological Chemistry2020ISSN 1083-351X
La, The Mon et al.Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytesFASEB Journal2020ISSN 1530-6860
Martinez, Pablo et al.TANGLED1 mediates microtubule interactions that may promote division plane positioning in maizeJournal of Cell Biology2020ISSN 1540-8140
Kappelmann-Fenzl, Melanie et al.Complex formation with monomeric α-tubulin and importin 13 fosters c-jun protein stability and is required for c-jun’s nuclear translocation and activityCancers2019ISSN 2072-6694
Perchey, R T et al.p27Kip1 regulates the microtubule bundling activity of PRC1… et Biophysica Acta (BBA …2018Article Link
Pellacani, Claudia et al.Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregationeLife2018ISSN 2050-084X
Renna, Luciana et al.TGNap1 is required for microtubule-dependent homeostasis of a subpopulation of the plant trans-Golgi networkNature Communications2018ISSN 2041-1723
Liang, Hong et al.The microtubule-associated protein IQ67 DOMAIN5 modulates microtubule dynamics and pavement cell shapePlant Physiology2018ISSN 1532-2548
Vasileva, Ekaterina et al.The role of microtubules in the regulation of epithelial junctionsTissue Barriers2018ISSN 2168-8370
Bajaj, Rakhi et al.KNL1 Binding to PP1 and Microtubules Is Mutually ExclusiveStructure2018ISSN 1878-4186
Becher, Alexander et al.The armadillo protein p0071 controls KIF3 motor transportJournal of Cell Science2017ISSN 1477-9137
Golub, Ognjen et al.Activation of discs large by aPKC aligns the mitotic spindle to the polarity axis during asymmetric cell divisioneLife2017ISSN 2050-084X
Zhang, Xiang et al.Palladin is a novel microtubule-associated protein responsible for spindle orientationScientific Reports2017ISSN 2045-2322
Sun, Tao et al.JIP1 and JIP3 cooperate to mediate TrkB anterograde axonal transport by activating kinesin-1Cellular and Molecular Life Sciences2017ISSN 1420-9071
Kesten, Christopher et al.In vitro Microtubule Binding Assay and Dissociation Constant EstimationBio-Protocol2016ISSN 2331--8325
Kim, Juri et al.Identification of a novel microtubule-binding protein in Giardia lambliaKorean Journal of Parasitology2016ISSN 1738-0006
Liu, Zengyu et al.Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in ArabidopsisDevelopmental Cell2016ISSN 1878-1551
Wang, Peixiang et al.RAB-10 Promotes EHBP-1 Bridging of Filamentous Actin and Tubular Recycling EndosomesPLoS Genetics2016ISSN 1553-7404
Colbert, Paul L. et al.EphrinB1: Novel microtubule associated protein whose expression affects taxane sensitivityOncotarget2015ISSN 1949-2553
Endler, Anne et al.A Mechanism for Sustained Cellulose Synthesis during Salt StressCell2015ISSN 1097-4172
Hamm, Matthew et al.Physiologically relevant factors influence tau phosphorylation by leucine-rich repeat kinase 2Journal of Neuroscience Research2015ISSN 1097-4547
Shih, Pu Yun et al.Cortactin-binding protein 2 increases microtubule stability and regulates dendritic arborizationJournal of Cell Science2014ISSN 1477-9137
Jiang, Hao et al.A Microtubule-Associated Zinc Finger Protein, BuGZ, Regulates Mitotic Chromosome Alignment by Ensuring Bub3 Stability and Kinetochore TargetingDevelopmental Cell2014ISSN 1534-5807
Lee, Dongmin et al.Enhanced expression and purification of inositol 1,4,5-trisphosphate 3-kinase A through use of the pCold1-GST vector and a C-terminal hexahistidine tag in Escherichia coliProtein Expression and Purification2014ISSN 1046-5928
Rajgor, Dipen et al.Mammalian microtubule P-body dynamics are mediated by nesprin-1Journal of Cell Biology2014ISSN 1540-8140
Kim, Juri et al.Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 proteinPLoS ONE2014ISSN 1932-6203
Kitagawa, Mayumi et al.Cdk1 coordinates timely activation of MKlp2 kinesin with relocation of the chromosome passenger complex for cytokinesisCell Reports2014ISSN 2211-1247
He, Ze et al.Cytoplasmic retention of a nucleocytoplasmic protein TBC1D3 by microtubule network is required for enhanced EGFR signalingPLoS ONE2014ISSN 1932-6203
Alev, Cantas et al.Proteomic approaches for the study of electrical synapses and associated protein-interaction complexesNeuromethods2013
Zhu, Mei et al.MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progressionJournal of Cell Biology2013ISSN 0021-9525
Urbé, Sylvie et al.Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome-and microtubule-associated functionsMolecular Biology of the Cell2012ISSN 1059-1524
Fugelstad, Johanna et al.Functional characterization of the pleckstrin homology domain of a cellulose synthase from the oomycete Saprolegnia monoicaBiochemical and biophysical research communications2012ISSN 1090--2104
Lee, D et al.Inositol 1, 4, 5-Trisphosphate 3-Kinase A Is a Novel Microtubule-associated ProteinJournal of Biological …2012Article Link
Roth, Daniela Martino et al.Mechanism of microtubule-facilitated "fast track" nuclear importJournal of Biological Chemistry2011ISSN 0021-9258
Azakir, Bilal A. et al.Dysferlin interacts with tubulin and microtubules in mouse skeletal musclePLoS ONE2010ISSN 1932-6203
Nishi, Mayuko et al.Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 GagFEBS Letters2009ISSN 0014-5793
Moshnikova, Anna et al.Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive functionBMC Research Notes2008ISSN 1756-0500
Sackett, Dan L. et al.Intracellular proadrenomedullin-derived peptides decorate the microtubules and contribute to cytoskeleton functionEndocrinology2008ISSN 0013-7227
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Ziegelbauer, Joseph et al.Transcription Factor MIZ-1 Is Regulated via Microtubule AssociationMolecular Cell2001ISSN 1097--2765

 

Question 1: Can cell lysates be used with this kit as the source of a test protein?

Answer 1:  Yes, cell lysates can be used as the source of the test protein for examining binding between tubulin monomers, polymers and the protein of interest.  However, Cytoskeleton does not recommend this as the purity and concentration of the protein will often be too low to interact with tubulin monomers and polymers.  Also, the lysates will contain additional accessory proteins and multiple phosphatases and proteases that can interfere or alter the interactions between tubulin and the test protein.  We recommend consulting these citations which used cell lysates with this kit: Monzo et al., 2005. Clues to CD2-associated protein involvement in cytokinesis. Mol. Biol. Cell. 16, 2891–2902; Moshnikova et al., 2008. Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function. BMC Research Notes. 1, 13.  We also recommend the following modifications: 

Use 4 x 15 cm plates of cells.  Wash and scrape cells with a PIPES-based buffer at 4°C.  Use 3 mls of the following buffer: 20 mM PIPES, pH 7.0, 2 mM MgCl2, 1 mM EGTA, 1 mM GTP, 0.5 mM fresh PMSF (a protease inhibitor that has only 30 min half-life in aqueous solutions), and 1:100 dilution of 100X protease inhibitor cocktail (Cat. # PIC02).  Collect the scraped cells and lyse the cells with sonication at 4°C with 5 pulses on medium for 15 secs each with a 1 min cool down in between each sonication burst.  Then centrifµge the lysate at 14,000 x g in a microfµge at 4°C for 20 min.  Measure the protein in the supernatant and proceed by mixing 2 to 5 mg/ml of extract with 0.5 mg/ml microtubules, as described in the kit.

 

Question 2: What is an appropriate buffer for the test protein?

Answer 2: The test protein must be in an appropriate buffer to allow attachment to microtubules.  Conditions which do not favor microtubule binding are: high salt (e.g. >50 mM Buffer or NaCl), high or low pH (i.e. pH of <6.0 or >9.0) and high calcium (greater than 0.1 mM calcium will depolymerize microtubules).  EGTA is included at 1mM to chelate excess calcium.

It is often advisable to include 5.0 mM Mg2+ in the buffer.  Appropriate buffers include: HEPES, PIPES and MES, all at 20 mM.  In some cases, the presence of ATP or GTP in the reaction may prevent MAP/microtubule association (e.g., kinesin or dynamin, respectively).  

 

 If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com