Anti-microtubule drug test
Anti-microtubule drug test
Anti-microtubule drug experiments are mainly used to (1) find new anticancer drugs (2) study the mechanism of action of anticancer drugs.
Operation method
Anti-microtubule drug test
Principle
Microtubule protein solution at 0 ~ 4 ℃ is colorless and transparent solution, when the temperature rises, or 37 ℃ insulation, tubulin polymerization to generate microtubules, followed by an increase in the turbidity of the solution, the degree of absorption (OD) rise, which can be used in the spectrophotometer, measured at 350 nm wavelength, according to the measured OD value of the insulation time for the graph, plotted in the "S" type. polymerization curve. On the contrary, put the polymerized microtubule solution in a water bath. The depolymerization curve can also be determined.
Materials and Instruments
Animal brain tissue Move I. Preparation of microtubule proteins Caveat 1. In the experiment of microtubule protein preparation, it is important to take fresh materials, and try to start the experiment within 1 h after the execution of animals. The brain tissue after cryogenic freezing should not be used, otherwise the yield will not be high and the activity will be low. 2. All the vessels should be clean to avoid calcium ion contamination. 3. During the homogenization process, try to avoid excessive foam production to reduce protein denaturation. 4. pH has a greater impact on the in vitro polymerization of microtubules, in the drug screening, pay attention to the pH of the drug solution can not be too high or too low, try to make the addition of drug solution, microtubule protein solution pH does not change significantly. 5. Take the frozen microtubule protein solution, quickly rinse the wall of the bottle with room temperature water to make it melt, put it into an ice bath, dilute it with cold MES buffer (without GTP or ATP) to the desired concentration (2~3 mg/ml), add GTP or ATP to 1.0 mmol/L . 6. Set the "0 point" at 350 nm spectrophotometrically with the microtubule protein solution immediately removed from the ice bath. 7. Hold the cuvette at 37°C and measure the OD value at different times (every 1-2 min). (Measure every 1~2 min, after 5 min can extend the interval of measurement), until 20~30 min for the end. 8. Put the colorimetric cup into an ice bath and measure the OD value at any time until the OD value no longer decreases. Common Problems 1. Anti-microtubule drugs are a broad-spectrum class of chemotherapeutic agents that affect spindle formation by acting on cellular microtubules and inhibit cellular mitosis. Anti-microtubule drugs are broad-spectrum chemotherapeutic agents that affect spindle formation and inhibit cell mitosis by acting on cell microtubules. Currently, the main commonly used anti-microtubule drugs are: paclitaxel, docetaxel, vincristine, vincristine and vincristine. 2. Paclitaxel/docetaxel can promote the polymerization of microtubules in tumor cells and stabilize the polymerized microtubules after its action on tumor cells, leading to the aggregation of a large number of microtubules in the cells, which in turn interferes with the various functions of the cells, especially stopping the cells from dividing. Vincristine/vincristine can inhibit the polymerization of microtubule proteins in tumor cells, inhibit the formation of spindle microtubules, and cause the nuclear division to stop at the middle stage of cell division, thus inhibiting the growth of tumor cells. For more product details, please visit Aladdin Scientific website.
MES EGTA MgCl2 Glycerin DMSO
Water bath Centrifuge Spectrophotometer
1. Reagents and stock solution
(1) MES reservoir solution (2N(-Morpholino)ethanesulfonic acid) 0.5 mol, MES 19.25 g dissolved in 200 ml of double-distilled water.
(2) EGTA reservoir solution [Ethylene-bis (oxyethylenitrile)] tetraacetic acid] 5 mmol, EGTA 90 mg dissolved in 100 ml of double-distilled water, can be completely dissolved with a small amount of 1 mol/L NaOH solution.
(3) MgCl2 storage solution 0.1 mol/L.
2. Buffer preparation
(1) MES buffer: adjust pH to 6.5 with 1 mol/L NaOH, add double-distilled water to 400 mL, store at 4°C and add GTP or ATP to 1 mmol before use.
(2) Microtubule polymerization buffer: adjust pH to 6.5 with 1 mol/L NaOH, add double-distilled water to 50 mL, store at 4°C, and add GTP or ATP to 1 mmol before use.
3. Sampling
Microtubules are commonly found in their nucleated cells, but are most abundant in animal brain tissue. Generally, pig brain and cow brain are taken because of their convenient source and cheap price. There are also rabbit, chicken, rat brain tissue and sea urchin eggs as materials.
4. Operation
(1) Take fresh animal brain tissue, strip off the meninges and large blood vessels, cut.
(2) Wash 1-2 times with cold MES buffer.
(3) Add MES buffer at a ratio of 0.5~1 mL per gram of brain tissue, and make homogenization with an electric (or manual) glass homogenizer with a Teflon mallet at 4℃.
(4) Centrifuge at 105,000 g for 1 h at 4°C and remove the supernatant.
(5) Add an equal volume of microtubule polymerization buffer, and keep warm at 37℃ for 30 min (shaking or gently stirring several times in the middle at the same time).
(6) Centrifuge at 26℃, 105,000 g for 1 h.
(7) Take the precipitate, add 1/10 homogenization volume of cold MES buffer, gently stir or use a homogenizer to crush the precipitate.
(8) Place the suspension on ice for 0.5 h to completely dissolve the precipitate. Repeat the low and high temperature centrifugation once more.
(9) After these two cycles, 85%~95% of the microtubule protein is produced, and the rest is microtubule-associated protein.
(10) Determination of protein content. Dilute to 4~5 mg/mL with MES buffer and store in liquid nitrogen for backup or further purification.Determination of microphotoprotein activity
1. MES buffer, prepared as above.
2. ATP (or GTP) solution 100 mmol/L stock solution, prepared on the day of the test, added to the microtubule solution before the test, with a final concentration of 1 mmol/L.
(1) Remove the frozen microtubule protein solution and quickly flush the walls of the bottle with room temperature water to melt it.
(2) Place in an ice bath, dilute with MES buffer to the desired concentration (2~3 mg/ml) and add ATP to 1 mmol/L.
(3) Remove the microtubule protein solution immediately from the ice bath and set it at "0" at 350 nm on a spectrophotometer.
(4) The colorimetry was then kept warm at 37°C in water and the OD values were measured at different times.
(5) At the beginning, it is better to measure every 1~2 min, and after 5 min, it can be measured at longer intervals.
(6) Until 20~30 min, put the colorimetric cup into the ice bath, at this time the polymerized microtubules and depolymerization, can be measured at any time until the OD value is no longer decreasing.(7) to the measured OD value on the insulation time graph can be obtained a positive "S" type polymerization curve, and inverted "S" type depolymerization curve (Figure 65-4) can be seen, 37 ℃ insulation began 1 ~ 2 min, is a short latency period, and then Then the curve showed an exponential increase.
(8) The ping value is reached after 5 min. When the temperature is changed to 0℃, the curve decreases rapidly, but the end of the depolymerization curve is slightly higher than the beginning, which may be related to a small amount of aggregates that cannot be depolymerized during polymerization.
(9) This polymerization curve indicates that microtubulin has good activity, and the ping value is proportional to the concentration of tubulin (Figure 65-5).
(10) The above method is easier, faster and more accurate if it is done on a spectrophotometer with a high or low temperature water bath and an automatic scanning recorder. 
III. Screening of anti-microtubule drugs
1. Specific operation
(1) If the drug or compound can be dissolved in water, use MES buffer to form a solution of 10 mmol/L. If it is not soluble in water, use ethanol, methanol, DMSO to dissolve it, and put it in the refrigerator.
(2) Generally from 0.1 mmol/L concentration to determine the effect of drugs on microtubules of the experiment, if found to have a significant effect, can be further diluted 3 ~ 5 concentrations for measurement.
(3) If no obvious effect is seen at 0.1 mmol/L, the drug is considered to have a weak effect on the microtubules and no further measurement is necessary.(4) The specific test method and steps are the same as before (determination of microtubule protein polymerization activity).
2. Judgment and calculation of results
(1) Example of drugs that inhibit microtubule polymerization: colchicine (Figure 65-6). The inhibition rate is calculated by taking the "OD" of each tube at 37℃ for 20 min according to the following formula: "OD" of each tube is calculated by taking the "OD" of each tube at 37℃ for 20 min. 
(2) Example of a drug that promotes polymerization and inhibits depolymerization: paclitaxel (Figure 65-7). As seen in Figure 65-7, after the addition of the drug.
① The latency period is shortened or even disappears.
② The shape of the polymerization curve is significantly changed compared with the control.
③ When the temperature was changed to 0-4 redundancy, the microtubules could not be completely depolymerized.
From the above changes, it is not difficult to determine that the drug has a significant effect on microtubules.
