Tubulin porcine brain, T240-DX——tubulin (pig brain, purity>99%) Tubulin was purified from porcine brain using the method of Shelanski et al., and further purified to a purity of more than 99% by cation exchange chromatography . Supply of white freeze-dried powder.
1. The purpose of Tubulin porcine brain
Mainly manifested in four aspects:
1. IC50 and EC50 determination of anti-tubulin ligand;
2. Microtubule integration research;
3. Tubulin monomer binding research;
4. HDAC6 research;
5. Microtubule-activated motor protein atp enzyme analysis.
2. Material composition of Tubulin porcine brain
Tubulin was purified from pig brain using the method of Shelanski et al. and further purified to a purity of more than 99% by cation exchange chromatography. Supply of white freeze-dried powder.
The product has complete polymerization activity and uses zhuanli technology for freeze-drying to improve stability and service life. T240 is stable for 1 year under dry conditions at 4°C. If your project requires the same batch of tubulin to obtain consistent results, it is strongly recommended that you purchase the product in batches to save time and money. This product can replace our high-purity bovine tubulin products (Cat. #TL238, T238 and T237) and work in the same way.
3. The purity of Tubulin porcine brain
Determined by protein scanning density on SDS-PAGE gel. The sample purity is >99%.
Fourth, the biological activity of Tubulin porcine brain
One unit of tubulin is defined as 5.0 mg purified protein (determined by the precision red advanced protein analysis reagent cat). #ADV02). The biological activity of T240 was evaluated by tubulin polymerization test. The ability of tubulin to polymerize into microtubules can be observed by observing the increase in the optical density of the tubulin solution at 340 nm. When using a spectrophotometer with a path length of 0.8 cm (the sample volume in a 1/2 area 96-well plate is 180 µl), a 5 mg/ml tubulin solution in a general tubulin buffer plus 5% glycerol and 1 mM GTP, an OD340 nm reading of 0.75-1.10 should be obtained within 30 minutes.
It should be noted that tubulin minus glycerol will not polymerize in G-PEM buffer until the tubulin concentration is very high (>10 mg/ml). Even at these concentrations, polymerization is relatively slow. In the case of a low concentration of tubulin-glycerol, high-efficiency polymerization can be achieved by adding a polymerization promoting compound (for example, glycerol, paclitaxel, or dimethyl sulfoxide).
Five, two questions about tubulin
Q1: What is the correct way to store tubulin to ensure maximum stability and activity?
A: The recommended storage conditions for tubulin freeze-dried products are 4°C, and a desiccant is added to keep the humidity below 10%. Under these conditions, the protein can be stable for 6 months. Lyophilized protein can also be stored dry at -70°C, where it can be stable for 6 months. However, at -70°C, the rubber seal on the tube cover may break and get moisture. Therefore, we recommend storing at 4°C. If stored at -70°C, if this storage condition is used, a desiccant must be added to the freeze-dried protein. After recombining the protein according to the instructions, aliquots of the concentrated protein in G-PEM buffer should be quickly frozen in liquid nitrogen and stored at -70°C (stable for 6 months). Note: It is very important to freeze tubulin quickly in liquid nitrogen, as other freezing methods can cause a significant decrease in activity. Thaw quickly in a room temperature water bath for 1 minute. Avoid repeated freeze-thaw cycles.
Q2: Why is the cytoskeleton recommended to use common tubulin buffer and GTP to restore tubulin?
A: In order to maintain the conformation of tubulin and tubulin, we recommend using GTP+tubulin monomer. For resuspension, we recommend using ordinary tubulin buffer (Cat. #BST01-001), including 80 mM tubing, 2 mM MgCl2, 1 mM EGTA, pH 7.0, supplemented with 1 mM GTP (Cat. #BST06-001) . Tubulin requires GTP and magnesium ions to maintain proper stability and conformation, even in a monomeric state. GTP is also required in the polymerization process, because its hydrolysis during tubulin polymerization is necessary for polymerization to occur. EGTA is a calcium chelator and an effective inhibitor of tubulin polymerization. Glycerol is usually added to a final concentration of 5-10% to enhance polymerization; however, glycerol is not necessary to maintain biologically active tubulin, and it is not necessary to include glycerol during reconstitution and storage of tubulin. When the recombinant tubulin is stored in batches, the tubulin must be aliquoted and quickly frozen in liquid nitrogen with a concentration greater than 6 mg/ml to maintain the biological activity of the tubulin. Then, the aliquot should be stored at -70°C. When thawing an aliquot, it should be quickly thawed in a room temperature water bath and placed on ice until the test is used.
Tubulin porcine brain link:
http://www.amyjet.com/products/T240-DX.shtml