Immunoscreening assay

Summary

A technique for selecting a target from a population based on the principle of antigen-antibody interaction. For example, antibodies are used to detect proteins synthesized from expressed gene libraries to screen for clones of the target gene.

Operation method

basic program

Materials and Instruments

cDNA
BHI chloramphenicol NaOH SSC chloroform isoamyl alcohol TES SDS mRNA methionine ethanol sodium acetate immunoprecipitation buffer
Rotor Nitric acid fiber filter membrane Centrifuge tube Micro porous washer

Move

1. Add 250 μl of selective BHI culture medium containing appropriate antibiotics to each well of the microtitre plate and inoculate each with an independent cDNA clone and incubate at 37°C overnight.

2. Add 50 ml of BHI/antibiotic culture solution to a 250 ml flask, and take 100 μl of culture solution from each microwell in groups of 10 clones overnight, mix and inoculate into the flask.

3. Incubate at 37°C until _A590=0.7, then add chloramphenicol and continue to incubate at 37°C overnight; repeat the incubation for each group of 10 overnight clones.

4. Plasmid DNA was prepared by centrifugation at 2 000 g 4°C for 10 min.5. Dissolve about 50 μg of the prepared plasmid DNA in 1.5 ml of TE buffer and transfer into a 15 ml sterile, capped glass culture tube.

6. Add 1.5 ml of 1 mol/l NaOH immediately after 10 min of boiling water bath and leave for 10 min at room temperature.

7. Add 9 ml of neutralizing solution, mix well and place in ice bath, test pH only should be between 6.5~7.5.8. A 0.45 μm pore size nitrocellulose filter membrane is placed on a porous filter connected to a vacuum pump.

9. Add the denatured DNA solution obtained in step 4 at a rate of 1 ml/min and continue pumping for 3 min after all liquid has been aspirated.

10. The membrane was removed and washed with 50 ml of 6×SSC, and then baked in a vacuum oven at 80°C for 2 h. The membrane was then dried in a vacuum oven for 2 h. The membrane was then dried in a vacuum oven at 80°C for 2 h.

11. Use a sterile single-hole punch to punch out small round pieces of membrane with a diameter of 5 mm on the filter membrane, and mark them with a ballpoint pen.12. Preheat 0.3 ml of hybridization solution containing 10-50 μg of poly(A) ⁺ RNA in a sterile capped plastic tube at 70°C for 10 min.

13. 10 pieces of small filter membranes were then placed in the hybridization solution and incubated at 50°C for 2 h. The membrane was then incubated for 2 h at 50°C.

14. Transfer the small filter membrane into 50 ml tubes (each tube can contain up to 20 pieces of membrane), each time with 225 ml TES/0.5% SDS Sin solution at 65 ℃ rotary shaking wash 30 s, a total of 10 times, and then aspirate off the upper please.

15. Then wash the membrane with 25 ml of TES solution at 65℃ for 2 times.

16. Transfer each membrane to a sterile, siliconized 1.5 ml microcentrifuge tube and add 0.3 ml of water and 2 μl of 10 mg/ml yeast tRNA to each tube.

17. Denature in a boiling water bath for 60 s. Immediately snap-freeze in dry ice or cold ethanol. Then thaw at room temperature.18. Remove the filter membrane with a sterile needle, add 150 μl of saturated phenol and 150 chloroform/isoamyl alcohol to each microcentrifuge tube, mix well, centrifuge and transfer the aqueous phase to a sterile microcentrifuge tube.

19. Add 30 μl of 3 mol/l sodium acetate and 2 times the volume of anhydrous ethanol, centrifuge to precipitate nucleic acids for 15 min, and lyophilize after washing once with 0.5 ml ethanol.

20. Resuspend this hybridization-selected RNA in 10 μl of water.

21. Take 5 μl of hybridization-selected RNA, add 10 μl of translation reaction mixture containing labeled methionine, and incubate at 30℃ for 60 min.22. Add 15 μl of immunoprecipitation buffer and 1 μl of unimmunized serum and incubate for 10 min at room temperature.

23. Add 40 μl of Protein A-Sepharose suspension and allow to stand at room temperature for 30 min, centrifuge for 2 min and transfer the supernatant to a new microcentrifuge tube.

24. Add 1 μl of polyclonal or monoclonal antibody against the gene product of interest and incubate for 10 min at room temperature.25. Add 40 μl of protein suspension and react for 30 min at room temperature, centrifuge and discard the supernatant.26. Clean the protein A-Sepharose beads with 1 ml of Immunoprecipitation Buffer, 1 ml of High Salt Immunoprecipitation Buffer and 1 ml of water.27. Resuspend the precipitate with 20 μl of 2 × SDS sample buffer and boil for 10 min.

28. Wash down the translation products adsorbed on Protein A-Sepharose microbeads, centrifuge and aliquot into 15-20 ml small portions for denaturing SDS-PAGE electrophoresis.29. Dry glue and radiographic self-development exposure.

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