Identification and characterization experiments of human tumor antigens

Summary

Human tumor antigens can be (1) used for tumor diagnosis and (2) immunotherapy.

Operation method

immunoprecipitation

Principle

In this experiment, immunocomplexes are purified using Dynabeads protein A. Dynabeads Protein A has the following effects on immunoprecipitation: (1) reduces protocol time to 30 minutes. (2) Significantly reduces the background caused by non-specific binding (3) Maintains the integrity of the protein complex. There is no physical stress on valuable proteins.

Materials and Instruments

Tumor cells
PBS physiological saline lysate protease inhibitors
Culture flasks Cell scrapers Centrifuge tubes Cryo-centrifuge -80°C refrigerator Probe-based ultrasound Constant temperature water baths

Move

I. Cell lysis
1. Scrape the cells in the direction. Transfer the cell suspension to a centrifuge tube, centrifuge the precipitate, and store at -80°C. (Collect cells quickly and at low temperatures to minimize the effect of protease). (Collect cells quickly and at low temperatures to minimize the effect of proteases)
2. Cell lysis. Use RIPA lysis system, pre-cooled at 40℃ before use, add 1.0 ml of lysate to ¹⁰⁷ cells, blow and mix well, add appropriate amount of protease inhibitor (e.g. cocktail), ice.
（Add appropriate amount of protease inhibitor (e.g. cocktail) and leave on ice for 3~5 minutes.
3. Sonicate the lysate. Use probe-type ultrasound to perform multiple short shocks of appropriate frequency, 10-20 seconds each, repeated 3 times with 10-20 seconds intervals, in an ice bath.
4. Centrifuge at 15 000 rpm, 40°C for 15 min and aspirate the supernatant in another EP tube on ice. The supernatant was used for the next pretreatment.Pretreatment of lysate
The lysates were pretreated with normal human serum.
1. Add normal human serum at the ratio of 1 ml lysate to 2 ul control serum.
2. Incubate at room temperature for 2-3 hours with slow shaking.
Purification of immune complexes
1. Use Dynabeads protein A for purification of immune complexes.2. Vibrate Dynabeads protein A and mix well. Pipette 100 ul of magnetic beads into an EP tube and place it on a magnet rack (Dynal MPC), the magnetic beads will be adsorbed onto the wall of the tube and the supernatant will be discarded.3. Add 500 ul of 0.1 M Na-phosphate (pH 8.1) to the Ep tube, gently rub the tube for about 2~3 min, place the EP tube on the magnet rack, the magnetic beads were adsorbed to the wall of the tube, and the supernatant was discarded.4. Repeat step 2 twice.5. After cleaning the magnetic beads, add 500 ul of pretreated lysate, shake the tube repeatedly, and react for 10~20 min at room temperature.6. Place the EP tube on the magnet rack, the magnetic beads are adsorbed to the wall of the tube, and transfer the supernatant to another EP tube.7. Wash the beads 3 times with RIPA lysate.8. Elute the antigen-antibody complex. Add 30 ul of 0.1 M citrate (pH 3.1) to the Ep tube, gently rub the tube for about 2~3 min, place the P tube on the magnet rack and aspirate the supernatant into an EP tube.9. Repeat step 7 and collect the supernatant in the same EP tube to elute a total volume of 60 ul of sample for control.10. The beads were washed three times with 0.1 M Na-phosphate (pH 8.1 ) and set aside.11. Add patient serum (1 ml with 2 ul of serum) to the supernatant retained in step 5, shake slowly and incubate for 2-3 h at room temperature.12. Place the EP tube on a magnet rack and attach the magnetic beads to the wall of the tube, discard the supernatant.13. Repeat steps 6~8. A total of two samples were collected, 60 ul each of purified immune complex from control and patient.14. The beads were washed three times with 0.1 M Na-phosphate (pH 8.1) and added to 100 ul of column buffer and stored at 40 °C.15. Add 2x SDS Sampling Buffer to the sample and adjust the pH with 1 M NaOH until Bromophenol Blue is blue.Analysis of results

1. 1D SDS-PAGE with immunoprecipitation can be used directly for one-dimensional gel electrophoresis or coupling agent modification of protein A or protein G on magnetic beads, and the eluted sample can be used for Western Blot. 2.

2. RIPA lysis solution:150 mmol/L NaCl; 1.0% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 50 mmol/L Tris (pH 8.0).

Caveat

To avoid co-elution of the antibody, the antibody should be crosslinked to the immunomagnetic beads before proceeding with the immunoprecipitation, and the use of a cross-linker is recommended.

Common Problems

The amount of Ig captured by Dynabeads protein A depends on the concentration of Ig in the starting sample. 100 µl of Dynabeads A protein isolates approximately 25-30 µg of human IgG from a sample containing 20-200 µg IgG ⁄ ml. The majority of sites bind to Fc for optimal Ig orientation.

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