1.Collect cells.Place the culture bottle on ice, pour out the culture medium, and rinse it with PBS or normal saline for two times.
Then leave some liquid in the bottle, and then use a special cell scraper to scrape cells in one direction.
Transfer the cell suspension to the centrifuge tube, centrifugate and take the precipitate, and store it at - 800°C.
2.Lysate cell.Ripa cracking system was used, pre cooled at 40C before use, and 107 cells were added 1.0ml lysate, blow and mix well, add a proper amount of protease inhibitor (such as cocktail), place on ice for 3-5 minutes.
3.Ultrasonic treatment of cracking liquid. Use probe type ultrasound for several short shocks of appropriate frequency, 10-20 second / time, repeat 3 times, the interval between 10-20 seconds, under the ice bath.
4.Centrifuge at 15000rpm and 4 ° C for 15min, draw the supernatant into another EP tube and place it on ice. upper the clear liquid is used for the next pretreatment.
The lysate was pretreated with normal human serum.
1.The normal human serum was added in the proportion of 1ml lysate and 2 μl control serum.
2.Incubate at room temperature for 2-3 hours and shake slowly.
1.The immune complex was purified by Dynabeads protein A.
2.Shake and mix Dynabeads protein A evenly, draw 100 μ l magnetic beads into EP tube, place them on the magnet frame, absorb the beads onto the tube wall, and discard the supernatant.
3.Add 500 μ l 0.1M Na Phosphorite (pH 8.1) to EP pipe, gently rub the pipe for about 2-3min, place EP pipe on the magnet frame, absorb the magnetic beads to the pipe wall, and discard the supernatant.
4.Repeat step 2 two times.
5.After cleaning the beads, add 500 μ l of pretreated lysate, shake the tube repeatedly and react at room temperature 10~20min.
6.The EP tube is placed on the magnet frame, the magnetic bead is adsorbed on the tube wall, and the supernatant is transferred to another EP tube.
7.The magnetic beads were cleaned three times with RIPA cracking solution.
8.Elute antigen antibody complex.Add 0.1M citrate (pH3.1) 30 μl into EP tube, gently rub tube for about 2-3min.Put the EP pipe on the magnet frame, draw and clean it on an EP pipe.
9.Repeat step 7, collect the supernatant in the same EP tube, elute the sample with a total volume of 60 μ L, and use it as a contrast.
10.The magnetic beads shall be cleaned with 0.1M Na-phosphor (pH 8.1) for three times and then standby.
11.Add patient's serum (1ml plus 2 μl serum) to the supernatant retained in step 5，slowly shake and incubate at room temperature for 2 ~ 3h.
12.Put the EP pipe on the magnet frame, and the magnetic bead will be adsorbed on the pipe wall, and then discard the supernatant.
13.Repeat steps 6-8.Two samples were collected, 60 μ L for the control and 60 μl for the patient respectively.
14.The magnetic beads were washed with 0.1M Na-phosphorite (pH 8.1) for three times and then added to the column storage solution for 100 μl and stored at 4℃.
15.Add 2 × SDS sample buffer to the sample and adjust the pH with 1 M NaOH to make Bromophenol blue blue.
The samples prepared by immunoprecipitation can be directly processed by one dimensional gel electrophoresis, or the protein A or protein G on the magnetic beads can be modified by coupling agent. The samples can be Western Blot after elution.