Microtubule/Tubulin In Vivo Assay Biochem Kit

Microtubule/Tubulin In Vivo Assay Biochem Kit
$0.00

Product Uses Include

  • To study the effects of pharmaceutical compounds on the ratio of Tubulin to Microtubules in cells.
  • To study the effects of mutated cell lines versus their parent cell line for the change in ratio of Tubulin to Microtubules.
  • To study the effects of physical alterations of environment on the ratio of Tubulin to Microtubules in cells.

Introduction
The most reproducible and accurate method of determining the amount of microtubule content versus free-tubulin content in a cell population is to use western blot quantitation of microtubule and free-tubulin cellular fractions.  The general approach is to homogenize cells in microtubule stabilization buffer, followed by centrifugation to separate the microtubules from free-tubulin pool. Then the fractions are separated by PAGE and tubulin is quantitated by western blot. The final result gives the most accurate method of determining the ratio of tubulin incorporated into the cytoskeleton versus the free-tubulin found in the cytosol. This kit contains all the reagents to perform this assay.

Kit contents
The kit contains sufficient materials for 30-100 assays depending assay setup and includes reagents for positive and negative controls. The following components are included:

  1. Lysis and Microtubule stabilization buffer
  2. GTP (Cat. # BST06)
  3. ATP (Cat. # BSA04)
  4. Protease inhibitor cocktail (Cat. # PIC02)
  5. Microtubule enhancing control solution
  6. Microtubule depolymerization control solution
  7. Control Tubulin Standard (Cat. # TL238)
  8. Tubulin monoclonal antibody
  9. SDS sample buffer (5 x)
  10. DMSO
  11. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. Centrifuge capable of temperature controlled operation at 100,000 x g with volumes of 100 µl to 2 ml depending on the cell lysis volume
  2. SDS-PAGE minigel system and western blotting transfer apparatus

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
Damuka, Naresh et al.Initial Evaluations of the Microtubule-Based PET Radiotracer, [11C]MPC-6827 in a Rodent Model of Cocaine AbuseFrontiers in Medicine2022ISSN 2296-858X
Guerra San Juan, Irune et al.Loss of mouse Stmn2 function causes motor neuropathyNeuron2022ISSN 0896-6273
Wu, Siwen et al.KIF15 supports spermatogenesis via its effects on Sertoli cell microtubule, actin, vimentin, and septin cytoskeletonsEndocrinology (United States)2021ISSN 1945-7170
Sun, Chiao Yin et al.LMBD1 protein participates in cell mitosis by regulating microtubule assemblyBiochemical Journal2021ISSN 1470-8728
Li, Huitao et al.NC1-Peptide from collagen α3 (IV) chains in the basement membrane of testes regulates spermatogenesis via p-FAK-Y407Endocrinology (United States)2020ISSN 1945-7170
Li, Linxi et al.Planar cell polarity protein Dishevelled 3 (Dvl3) regulates ectoplasmic specialization (ES) dynamics in the testis through changes in cytoskeletal organizationCell Death and Disease2019ISSN 2041-4889
Su, Wenhui et al.Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganizationFASEB journal : official publication of the Federation of American Societies for Experimental Biology2019ISSN 1530-6860
Eberle-Singh, Jaime A. et al.Effective delivery of a microtubule polymerization inhibitor synergizes with standard regimens in models of pancreatic ductal adenocarcinomaClinical Cancer Research2019ISSN 1557-3265
Mao, Bai Ping et al.CaMSAP2 is a microtubule minus-end targeting protein that regulates BTB dynamics through cytoskeletal organizationEndocrinology2019ISSN 1945-7170
Wen, Qing et al.Myosin VIIa supports spermatid/organelle transport and cell adhesion during spermatogenesis in the rat testisEndocrinology2019ISSN 1945-7170
Li, Lin Xi et al.Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex: An in vitro studyAsian Journal of Andrology2019ISSN 1745-7262
Wen, Qing et al.Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testisCell Death and Disease2018ISSN 2041-4889
Wen, Qing et al.Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testisAmerican Journal of Physiology - Endocrinology and Metabolism2018ISSN 1522-1555
Zhang, Guanyi et al.Androgen receptor splice variants circumvent AR blockade by microtubule-targeting agentsOncotarget2015ISSN 1949-2553
Niesman, Ingrid R. et al.Caveolin isoform switching as a molecular, structural, and metabolic regulator of microgliaMolecular and Cellular Neuroscience2013ISSN 1044-7431
Kapur, Mridu et al.Calcium tips the balance: A microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4EMBO Reports2012ISSN 1469-221X
Fan, Jianguo et al.A role for γs-crystallin in the organization of actin and fiber cell maturation in the mouse lensFEBS Journal2012ISSN 1742-464X
Roth, Daniela Martino et al.Mechanism of microtubule-facilitated "fast track" nuclear importJournal of Biological Chemistry2011ISSN 0021-9258
Mourad, Nizar I. et al.Metabolic amplification of insulin secretion by glucose is independent of β-cell microtubulesAmerican journal of physiology. Cell physiology2011ISSN 1522--1563
Liu, Jian J. et al.A mechanism of Rap1-induced stabilization of endothelial cell-cell junctionsMolecular Biology of the Cell2011ISSN 1059-1524
Romero, Ana M. et al.Chronic ethanol exposure alters the levels, assembly, and cellular organization of the actin cytoskeleton and microtubules in hippocampal neurons in primary cultureToxicological Sciences2010ISSN 1096-6080
Moseley, Gregory W. et al.Dual modes of rabies P-protein association with microtubules: A novel strategy to suppress the antiviral responseJournal of Cell Science2009ISSN 0021-9533
Rayala, Suresh K. et al.Dynamic interplay between nitration and phosphorylation of tubulin cofactor B in the control of microtubule dynamicsProceedings of the National Academy of Sciences of the United States of America2007ISSN 1091-6490
Roth, Daniela Martino et al.A microtubule-facilitated nuclear import pathway for cancer regulatory proteinsTraffic2007ISSN 1398-9219
Reiter-Funk, Cindy K. et al.Chronic ethanol exposure increases microtubule content in PC12 cellsBMC Neuroscience2005ISSN 1471-2202
Davis, Francesca J. et al.Concurrent opposite effects of trichostatin A, an inhibitor of histone deacetylases, on expression of α-MHC and cardiac tubulins: Implication for gain in cardiac muscle contractilityAmerican Journal of Physiology - Heart and Circulatory Physiology2005ISSN 0363-6135
Vogl, Thomas et al.MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytesBlood2004ISSN 0006-4971

 

Question 1:  When lysing the cells, how do I prevent existing tubulin monomers from polymerizing onto existing microtubules?

Answer 1:  The microtubules/tubulin in vivo assay requires a constant cells-to-buffer volume ratio.  Essentially the lysis step has to dilute the cellular extract so that the free tubulin does not polymerize onto existing microtubules (MTs). This ratio is roµghly 10 volumes of buffer to 1 volume of cell pellet.  Larger volumes of buffer are fine and in this kit the ratio is targeted at 50 volumes of buffer per volume of cells.  Additionally, the average cell size is important in designing the experiment, so be sure to estimate the average cell size of your culture so that you can use it to calculate a good estimate for the volume of lysis buffer required.

 

Question 2:  Is it possible to quantify the absolute amount of total tubulin in each of the experimental samples?

Answer 2:  Absolute quantitation of cellular tubulin can be performed using the tubulin standard as a positive control at 50, 20, 10, 5 and 2 ng per lane.

 

 

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