First, homogenate medium
Generally, 0.05 mol/L Tris-HCl, pH 7.4 phosphate buffer (PBS) is used. The customer can set the concentration according to the sample and the measurement index, in order to maintain the isotonic environment of the sample.
Second, the preparation of tissue homogenate
1. Take the tissue block (0.2g ~ 0.5g) at least 5 ~ 10mg in chilled PBS rinse, filter paper dried, accurately weighed, placed in a 5ml homogenate tube.
2. Add 4 volumes of homogenized medium to the homogenate tube according to the weight (g): volume (ml) = 1:4. Under ice water bath conditions, the tissue block is cut as soon as possible with an ophthalmic small scissors.
3, the way of homogenization: manual homogenization, machine homogenization.
① Manual homogenization: insert the lower end into the vessel containing the mixture of ice water and water in the left hand. Insert the mast into the casing vertically by the right hand, rotate it up and down for dozens of times (6-8 minutes), fully grind and make. Into a 20% homogenate.
② Machine homogenization: 20% tissue homogenate is prepared by grinding the tissue masher 10000～15000 rpm, or it can be prepared by the internal cutting homogenizer (homogenization time 10 sec/time, gap 30 sec, continuous) 3 to 5 times, in ice water, the temperature can be extended appropriately)
Microscopic examination: Take a small amount of tissue homogenate for smear (direct smear, staining can be), observe whether the cells are broken under the microscope, if not, extend the homogenization time.
4. Prepare the prepared 20% homogenate by using a common centrifuge or a low-temperature low-speed centrifuge at 4000 rpm, centrifuge for 10 to 15 minutes, and take the supernatant for determination.
Third, the sample is saved:
Plant tissue samples are not measured for a while, and can be cryopreserved immediately. The lower the temperature, the better. If the temperature is not repeated in the middle, it can be stored for three months below -20 °C, and can be stored for six months below -70 °C.
The prepared homogenate is not recommended to be frozen. It is best to measure it on the same day. If the time is too long, the enzyme activity will decrease. Some indicators can be stored for 4 to 5 days at 4 °C (such as SOD for 2 to 3 days, MDA) Can store 3 to 5, total protein can be stored for 5 to 7 days, etc.)