Product Uses Include
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 is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >99% pure.
Figure 1: 100 µg of T238P was run on a 10% SDS-PAGE gel and stained with Coomassie Blue.
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 datasheet 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.
Shelanski, M. L., et al. (1973). Proc. Natl. Acad. Sci. USA. 70, 765-768
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|Chen, Jiayi et al.||Α-Tubulin Tail Modifications Regulate Microtubule Stability Through Selective Effector Recruitment, Not Changes in Intrinsic Polymer Dynamics||Developmental Cell||2021||ISSN 1878-1551|
|Qiu, Rongde et al.||Dynein activation in vivo is regulated by the nucleotide states of its AAA3 domain||Current Biology||2021||ISSN 1879-0445|
|Karki, Menuka et al.||A cytoskeletal function for PBRM1 reading methylated microtubules||Science Advances||2021||ISSN 2375-2548|
|von Appen, Alexander et al.||LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation||Nature||2020||ISSN 1476-4687|
|Zehr, Elena A. et al.||Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever Microtubules||Developmental Cell||2020||ISSN 1878-1551|
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|Fees, Colby P. et al.||A unified model for microtubule rescue||Molecular Biology of the Cell||2019||ISSN 1939-4586|
|Richard McIntosh, J. et al.||Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments||Journal of Cell Biology||2018||ISSN 1540-8140|
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|Yang, Jianhong et al.||Pironetin reacts covalently with cysteine-316 of α-tubulin to destabilize microtubule||Nature Communications||2016||ISSN 2041-1723|
|Yi, Jun Mei et al.||Dual targeting of microtubule and topoisomerase II by α-carboline derivative YCH337 for tumor proliferation and growth inhibition||Oncotarget||2015||ISSN 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 cells||Biochemical Pharmacology||2015||ISSN 1873-2968|
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|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 Cells||The Journal of Cell Biology||1998||ISSN 0021-9525|
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|Sosa, Hernando et al.||A Model for the Microtubule-Ncd Motor Protein Complex Obtained by Cryo-Electron Microscopy and Image Analysis||Cell||1997||ISSN 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.
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