Product Uses Include
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.
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:
|Damuka, Naresh et al.||Initial Evaluations of the Microtubule-Based PET Radiotracer, [11C]MPC-6827 in a Rodent Model of Cocaine Abuse||Frontiers in Medicine||2022||ISSN 2296-858X|
|Guerra San Juan, Irune et al.||Loss of mouse Stmn2 function causes motor neuropathy||Neuron||2022||ISSN 0896-6273|
|Wu, Siwen et al.||KIF15 supports spermatogenesis via its effects on Sertoli cell microtubule, actin, vimentin, and septin cytoskeletons||Endocrinology (United States)||2021||ISSN 1945-7170|
|Sun, Chiao Yin et al.||LMBD1 protein participates in cell mitosis by regulating microtubule assembly||Biochemical Journal||2021||ISSN 1470-8728|
|Li, Huitao et al.||NC1-Peptide from collagen α3 (IV) chains in the basement membrane of testes regulates spermatogenesis via p-FAK-Y407||Endocrinology (United States)||2020||ISSN 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 organization||Cell Death and Disease||2019||ISSN 2041-4889|
|Su, Wenhui et al.||Cdc42 is involved in NC1 peptide-regulated BTB dynamics through actin and microtubule cytoskeletal reorganization||FASEB journal : official publication of the Federation of American Societies for Experimental Biology||2019||ISSN 1530-6860|
|Eberle-Singh, Jaime A. et al.||Effective delivery of a microtubule polymerization inhibitor synergizes with standard regimens in models of pancreatic ductal adenocarcinoma||Clinical Cancer Research||2019||ISSN 1557-3265|
|Mao, Bai Ping et al.||CaMSAP2 is a microtubule minus-end targeting protein that regulates BTB dynamics through cytoskeletal organization||Endocrinology||2019||ISSN 1945-7170|
|Wen, Qing et al.||Myosin VIIa supports spermatid/organelle transport and cell adhesion during spermatogenesis in the rat testis||Endocrinology||2019||ISSN 1945-7170|
|Li, Lin Xi et al.||Regulation of blood-testis barrier dynamics by the mTORC1/rpS6 signaling complex: An in vitro study||Asian Journal of Andrology||2019||ISSN 1745-7262|
|Wen, Qing et al.||Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis||Cell Death and Disease||2018||ISSN 2041-4889|
|Wen, Qing et al.||Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis||American Journal of Physiology - Endocrinology and Metabolism||2018||ISSN 1522-1555|
|Zhang, Guanyi et al.||Androgen receptor splice variants circumvent AR blockade by microtubule-targeting agents||Oncotarget||2015||ISSN 1949-2553|
|Niesman, Ingrid R. et al.||Caveolin isoform switching as a molecular, structural, and metabolic regulator of microglia||Molecular and Cellular Neuroscience||2013||ISSN 1044-7431|
|Kapur, Mridu et al.||Calcium tips the balance: A microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4||EMBO Reports||2012||ISSN 1469-221X|
|Fan, Jianguo et al.||A role for γs-crystallin in the organization of actin and fiber cell maturation in the mouse lens||FEBS Journal||2012||ISSN 1742-464X|
|Roth, Daniela Martino et al.||Mechanism of microtubule-facilitated "fast track" nuclear import||Journal of Biological Chemistry||2011||ISSN 0021-9258|
|Mourad, Nizar I. et al.||Metabolic amplification of insulin secretion by glucose is independent of β-cell microtubules||American journal of physiology. Cell physiology||2011||ISSN 1522--1563|
|Liu, Jian J. et al.||A mechanism of Rap1-induced stabilization of endothelial cell-cell junctions||Molecular Biology of the Cell||2011||ISSN 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 culture||Toxicological Sciences||2010||ISSN 1096-6080|
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|Vogl, Thomas et al.||MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes||Blood||2004||ISSN 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.
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