1. Take the tissue block (0.2g~1g) to a minimum of 2~5mg, rinse in ice-cold normal saline, remove the blood, dry the filter paper, weigh it, and put it into a small beaker of 5 or 10ml.
2. Use a pipette to measure the pre-cooled homogenization medium (ph7.4, 0.01 mol/L Tris-HCL, 0.0001 MOL/LEDTA-2Na, 0.01 mol/L sucrose 0.8% sodium chloride solution) or 0.86. % cold saline, the total volume of homogenate medium or physiological saline should be 9 times the weight of the tissue block. Use a pipette or pipette to take a total of 2/3 of the homogenate medium or physiological saline in the beaker. Cut the tissue block as soon as possible with an ophthalmic small scissors (naturally, the operation should be carried out in an ice water bath, and the small beaker with tissue should be placed in ice water).
3, there are a variety of ways of homogenization: manual homogenization, machine homogenization, ultrasonic homogenization, repeated freezing and thawing.
Manual homogenization: Pour the shredded tissue into a glass homogenate tube, and then rinse the remaining tissue in the beaker with the remaining 1/3 homogenization medium or physiological saline, and pour it into the homogenate tube for homogenization. The left hand-held homogenization tube is inserted into the vessel containing the ice-water mixture, and the right hand is inserted into the casing vertically, rotated up and down for several dozen times (6-8 minutes), and fully ground to homogenize the tissue. .
Manual homogenization: Pour the shredded tissue into a glass homogenate tube, and then rinse the remaining tissue in the beaker with the remaining 1/3 homogenization medium or physiological saline, and pour it into the homogenate tube for homogenization. The left hand-held homogenization tube is inserted into the vessel containing the ice-water mixture, and the right hand is inserted into the casing vertically, rotated up and down for several dozen times (6-8 minutes), and fully ground to homogenize the tissue. .
Machine homogenization: 10% tissue homogenate was prepared by grinding with a tissue grinder (OMNIBead Ruptor 24 multi-sample grinding bead homogenizer) at 6 m/s or with an internal tissue homogenizer (Omni Prep multi-sample homogenizer) The homogenization time is 20 seconds/time, the gap is 10 seconds, and it is 3-5 times in succession.
Ultrasonic pulverization: crushing with an ultrasonic cell disrupter (OMNI Sonic Ruptor 400W), which can be disrupted by sonicating for 30 seconds at an amplitude of 12.7 mm.
Ultrasonic pulverization: crushing with an ultrasonic cell disrupter (OMNI Sonic Ruptor 400W), which can be disrupted by sonicating for 30 seconds at an amplitude of 12.7 mm.
Repeated freezing and thawing: cultured or isolated cells can be homogenized by the above method, or can be repeatedly frozen and dissolved for about 3 times (that is, let the cells add appropriate amount of hypotonic liquid or double distilled water to freeze in the low temperature refrigerator, dissolve, and then re-knot Ice, redissolve, repeated about 3 times), but some enzyme activity will be affected.
Take a small amount of tissue homogenate for smear (direct smear, stain can be), observe whether the cells are worn under the microscope, if not broken, you can extend the homogenization time or increase the number of freeze-thaw cycles.
Take a small amount of tissue homogenate for smear (direct smear, stain can be), observe whether the cells are worn under the microscope, if not broken, you can extend the homogenization time or increase the number of freeze-thaw cycles.
4. Centrifuge the prepared 10% homogenate in a normal centrifuge or low-temperature low-speed centrifuge at 3000r/min for 10-15 minutes. If ATPase is detected, it only needs 1000 rpm, centrifuge for 5 minutes, and centrifuge. The homogenate leaves the supernatant and discards the precipitate below.
According to your experimental needs, take appropriate amount of supernatant to carry out various measurements.
According to your experimental needs, take appropriate amount of supernatant to carry out various measurements.
This experimental protocol uses Omni's Bead Ruptor 24 multi-sample grinding bead homogenizer and Prep multi-sample homogenizer (provided by Beijing Pingliyang Co., Ltd.).
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