Tubulin protein (>99% pure): porcine brain

Tubulin protein (>99% pure): porcine brain
$0.00

Product Uses Include

  • IC50 & EC50 determinations for anti-tubulin ligands.
  • Microtubule binding studies
  • Tubulin monomer binding studies
  • HDAC6 studies
  • Microtubule activated kinesin ATPase assays

Material
Tubulin protein has been purified from porcine brain by an adaptation of the method of Shelanski et al. (1), Further purification to >99% purity was achieved by cation exchange chromatography. Tubulin is supplied at 10 mg/ml as a frozen liquid in G-PEM (General tubulin buffer (Cat. # BST01) with 1 mM GTP (Cat. # BST06)).

This product is also available in a lyophilized, fully active format (Cat. # T240). The lyophilized format tubulin has many advantages over the frozen format. T240 costs less and since it allows for ambient temperature shipping, shipping is also significantly less costly. In addition, the lyophilized format allows for simpler (+4°C desiccated vs -70°C) and longer storage.

T238P is simular to T237 when 10% glycerol is added. >99% pure tubulin is also available in a convenient lyophilized pre-formed microtubule format (Cat. # MT001) for use in e.g. kinesin ATPase assays and microtubule binding studies.

Purity
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >99% pure.

t238

Figure 1: 100 µg of T238P was run on a 10% SDS-PAGE gel and stained with Coomassie Blue. 

Biological Activity

The biological activity of T238P is assessed by a tubulin polymerization assay. The ability of tubulin to polymerize into microtubules is followed by observing an increase in optical density at 340nm (OD340 nm) of a tubulin solution. Under the experimental conditions defined below a 5 mg/ml tubulin solution in General tubulin buffer (Cat. # BST01) buffer plus 5% glycerol (Cat. # BST05) and 1 mM GTP (Cat. # BST06) should achieve an OD340 nm absorption reading between 0.75 - 1.10 in 30 min at 37°C. The assay volume is 180 ul and assumes a spectrophotometer pathlength of 0.8 cm. See Fig 2 on the TL238 product page for an example of tubulin polymerization results.


It should be noted that tubulin minus glycerol WILL NOT polymerize in G-PEM buffer until very high tubulin concentrations (>10 mg/ml). Even at these concentrations polymerization is comparatively slow. Efficient polymerization at lower concentration of tubulin minus glycerol can be achieved by addition of a polymerization stimulating compound, e.g.paclitaxel or DMSO. See Figure 2 on the T237 product page for polymerization in the presence of glycerol and paclitaxel.

References

Shelanski, M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768

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
Chen, Jiayi et al.Α-Tubulin Tail Modifications Regulate Microtubule Stability Through Selective Effector Recruitment, Not Changes in Intrinsic Polymer DynamicsDevelopmental Cell2021ISSN 1878-1551
Qiu, Rongde et al.Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domainCurrent Biology2021ISSN 1879-0445
Karki, Menuka et al.A cytoskeletal function for PBRM1 reading methylated microtubulesScience Advances2021ISSN 2375-2548
von Appen, Alexander et al.LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformationNature2020ISSN 1476-4687
Zehr, Elena A. et al.Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever MicrotubulesDevelopmental Cell2020ISSN 1878-1551
Gudimchuk, Nikita B. et al.Mechanisms of microtubule dynamics and force generation examined with computational modeling and electron cryotomographyNature Communications2020ISSN 2041-1723
Fees, Colby P. et al.A unified model for microtubule rescueMolecular Biology of the Cell2019ISSN 1939-4586
Richard McIntosh, J. et al.Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilamentsJournal of Cell Biology2018ISSN 1540-8140
Zhang, Kai et al.Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and ActivatedCell2017ISSN 1097-4172
Miyake, Yasuyuki et al.Structural insights into HDAC6 tubulin deacetylation and its selective inhibitionNature Chemical Biology2016ISSN 1552-4469
Yang, Jianhong et al.Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubuleNature Communications2016ISSN 2041-1723
Yi, Jun Mei et al.Dual targeting of microtubule and topoisomerase II by α-carboline derivative YCH337 for tumor proliferation and growth inhibitionOncotarget2015ISSN 1949-2553
Yan, Jun et al.A novel synthetic compound exerts effective anti-tumour activity in vivo via the inhibition of tubulin polymerisation in A549 cellsBiochemical Pharmacology2015ISSN 1873-2968
Volkov, Vladimir A. et al.Preparation of segmented microtubules to study motions driven by the disassembling microtubule endsJournal of Visualized Experiments2014ISSN 1940-087X
Szyk, Agnieszka et al.Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferaseCell2014ISSN 1097-4172
King, S. J. et al.Dynactin increases the processivity of the cytoplasmic dynein motorNature cell biology2000ISSN 1465--7392
Gradin, Helena Melander et al.Regulation of Microtubule Dynamics by Extracellular Signals: cAMP-dependent Protein Kinase Switches Off the Activity of Oncoprotein 18 in Intact CellsThe Journal of Cell Biology1998ISSN 0021-9525
Lee Virginia, M. Y. et al.Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17Science (New York, N.Y.)1998ISSN 0036--8075
Sosa, Hernando et al.A Model for the Microtubule-Ncd Motor Protein Complex Obtained by Cryo-Electron Microscopy and Image AnalysisCell1997ISSN 0092--8674

 

Question 1: Under what conditions should I use tubulin prepared as a frozen liquid vs lyophilized powder?

Answer 1:  Under most experimental conditions, we recommend using the lyophilized tubulin (Cat. # T240) as it offers improved storage and stability compared to the frozen liquid (Cat. # T238P) which must be stored at -70°C.  Some researchers favor the frozen tubulin stock for high resolution microscopy studies and crystallography work.

 

Question 2: Are there special instructions for thawing and aliquoting Cat. # T238P ?

Answer 2:  It is recommended that the frozen liquid tubulin (Cat. # T238P) be stored at -70°C, where it is stable for 6 months from the date of purchase. To use, the protein should be rapidly thawed in a room temperature water bath, immediately transferred to ice and aliquoted into “experiment sized” amounts. Snap freeze aliquots in liquid nitrogen and store at -70°C. Aliquots of T238P must be snap frozen in liquid nitrogen prior to storage at -70°C. Failure to do this results in significant loss of activity. Tubulin will be active for only 1 week if stored at -40°C.

 

 

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