Product Uses Include
Porcine brain tubulin (>99% pure, see Cat. # T240) has been modified so that random surface lysines contain a covalently linked, long-chain biotin derivative. A long-chain biotin derivative was selected for this procedure because it allows the biotin molecules to be spaced far enough away from the tubulin protein so as not to interfere with its activity, e.g., ligand binding to SPA beads or other streptavidin based reagents. Biotin labeled tubulin is supplied as a lyophilized powder.
Cytoskeleton, Inc. also offers biotinylated cancer cell tubulin (Cat. # H003 )
The protein purity of the tubulin used for labeling is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. The protein used for T333P is >99% pure tubulin. Labeled protein is run on an SDS gel, transfered to a nitrocellulose membrane and detected with streptavidin alkaline phosphatase (Fig 1). 10 ng of T333P is readily detectable. No free biotin is detectable in the final product.
Figure 1: Purity determination of biotin tubulin. 10 and 100 ng of T333P was run on a 4-20% SDS-PAGE gel, transferred to a nitrocellulose membrane and biotin labeled material was detected with streptavidin alkaline phosphatase.
The biological activity of T333P is assessed by a tubulin polymerization assay. To pass quality control, a 5 mg/ml solution of fluorescein labeled tubulin in G-PEM plus 5% glycerol must polymerize to >85%. This is comparable to unlabeled tubulin under identical conditions.
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|Zheng, Pengli et al.||ER proteins decipher the tubulin code to regulate organelle distribution||Nature||2022||ISSN 1476-4687|
|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|
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|Zhernov, Ilia et al.||Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional Diversity||Current Biology||2020||ISSN 1879-0445|
|Rodríguez-García, Ruddi et al.||Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules||Current Biology||2020||ISSN 1879-0445|
|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|
|Gaska, Ignas et al.||The Mitotic Crosslinking Protein PRC1 Acts Like a Mechanical Dashpot to Resist Microtubule Sliding||Developmental Cell||2020||ISSN 1878-1551|
|Adriaans, Ingrid E. et al.||MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during Anaphase||Current Biology||2020||ISSN 1879-0445|
|Peña, Alejandro et al.||Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy||Structure||2020||ISSN 1878-4186|
|Aher, Amol et al.||CLASP Mediates Microtubule Repair by Restricting Lattice Damage and Regulating Tubulin Incorporation||Current Biology||2020||ISSN 1879-0445|
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|Nakos, Konstantinos et al.||Septin 2/6/7 complexes tune microtubule plus-end growth and EB1 binding in a concentration- And filament-dependent manner||Molecular Biology of the Cell||2019||ISSN 1939-4586|
|Hu, Yuhan et al.||Regulation of MT dynamics via direct binding of an Abl family kinase||Journal of Cell Biology||2019||ISSN 1540-8140|
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|Fan, Yuanwei et al.||The Arabidopsis SPIRAL2 Protein Targets and Stabilizes Microtubule Minus Ends||Current Biology||2018||ISSN 0960-9822|
|Karasmanis, Eva P. et al.||Erratum: Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin (Developmental Cell (2018) 46(2) (204–218.e7), (S1534580718304982) (10.1016/j.devcel.2018.06.013))||Developmental Cell||2018||ISSN 1878-1551|
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|Maciejowski, John et al.||Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation||Developmental Cell||2017||ISSN 1878-1551|
|Arellano-Santoyo, Hugo et al.||A Tubulin Binding Switch Underlies Kip3/Kinesin-8 Depolymerase Activity||Developmental Cell||2017||ISSN 1878-1551|
|Kandel, Mikhail E. et al.||Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy||ACS Nano||2017||ISSN 1936-086X|
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Question 1: What is the proper way to store the tubulin to insure maximum stability and activity?
Answer 1: The recommended storage condition for the lyophilized tubulin product is 4°C with desiccant to maintain humidity at <10% humidity. Under these conditions the protein is stable for 6 months. Lyophilized protein can also be stored desiccated at -70°C where it will be stable for 6 months. However, at -70°C the rubber seal in the lid of the tube could crack and allow in moisture. Therefore we recommend storing at 4°C. If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized. After reconstituting the protein as directed, the concentrated protein in G-PEM buffer should be aliquoted, snap frozen in liquid nitrogen and stored at -70°C (stable for 6 months). NOTE: It is very important to snap freeze the tubulin in liquid nitrogen as other methods of freezing will result in significantly reduced activity. Defrost rapidly by placing in a room temperature water bath for 1 min. Avoid repeated freeze/thaw cycles.
Question 2: Does the biotinylated tubulin polymerize as well as unlabeled tubulin?
Answer 2: Yes, the biotinylated tubulin (Cat. # T333P) polymerizes as well as unlabeled tubulin (Cat. # 240). The biological activity of biotinylated tubulin is determined from its ability to efficiently polymerize into microtubules in vitro and separate from unpolymerized protein in a spin-down assay. Stringent quality control ensures that >85% of the biotinylated tubulin can polymerize in this assay. This is comparable to the polymerization capacity of unmodified tubulin (Cat. # T240).
Question 3: Why does Cytoskeleton recommend the use of general tubulin buffer and GTP for resuspending tubulin?
Answer 3: We recommend resuspending tubulin in general tubulin buffer + GTP to maintain tubulin monomer protein stability and conformation and to provide the necessary components for polymerization. For resuspension, we recommend using a general tubulin buffer (Cat. # BST01-001) which consists of 80 mM PIPES, 2 mM MgCl2, 1 mM EGTA, pH 7.0, supplemented with 1 mM GTP (Cat. # BST06-001). Tubulin requires GTP and magnesium ions for proper stability and conformation, even in its monomeric state. GTP is also required for the polymerization process as its hydrolysis during tubulin polymerization is necessary for polymerization to occur. EGTA is a chelator of calcium which is a potent inhibitor of tubulin polymerization. Glycerol is often added to a final concentration of 5 - 10% to enhance polymerization; however, glycerol is not necessary for the maintenance of biologically active tubulin and does not need to be included when reconstituting and storing tubulin. When aliquoting reconstituted tubulin for storage, it is essential to aliquot and snap-freeze tubulin in liquid nitrogen at a concentration of >6 mg/ml to preserve tubulin’s biological activity. Then the aliquots should be stored at -70°C. When thawing the aliquots, thaw rapidly in a room temperature water bath and place on ice until right before experimental use.
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