Escherichia coli Immunoprecipitations
1. Start minimal o/ns of the appropriate strains. A minimal unsupplemented E. coli o/n will take 20 hrs for saturated growth. Since this can make experimental planning inconvenient the minimal media can be supplemented with either LB added to 0.5% or a low sulphur amino acid mix added at 1X (see Appendix I for 10X low sulphur amino acid mix). A supplemented E. coli o/n will take 14 hrs for saturated growth.
2. Make 1/50 dilutions of the o/ns into fresh minimal media. The low sulphur amino acid mix can be added at 1X to increase the growth rate if desired. (Do not add LB as a supplement or labelling will not occur.)
3. Let the cultures grow until the A600 reaches 0.4 to 0.5.
4. The cells are labelled by adding 10 uCi of 35S methionine per 1 ml of culture. (The resulting antigen extract prepared from a 1 ml culture labelled with 10 uCi of 35S methionine is sufficient for one immunoprecipitation.) Labelling times (pulses) vary from 15 seconds to 1 minute depending on experimental conditions.
5. (Optional) - If a chase is desired after the initial 35S methionine pulse a 5% solution of cold L-methionine is added to the culture at final concentration of 0.5%. Chase times vary from 10 seconds to 1 minute depending on experimental conditions.
6. To stop the labelling, add trichloroacetic acid to the culture at a final concentration of 5%, vortex rapidly and place the culture on ice. Keep the trichloroacetic acid treated cultures on ice for 10 minutes to allow for complete precipitation.
7. Transfer the cultures to microfuge tubes and spin in a cold microfuge for 15 minutes.
8. Aspirate off the supernatant and wash twice with ice cold acetone. A
4 minute cold microfuge spin is sufficient to repellet the precipitated material during the acetone washes.
9. Dry the samples in the speed vac for 5 to 10 minutes.
10. Add 50 ul of boiling buffer (10 mM Tris-Cl: pH 7.5, 1 mM EDTA; pH 8.0, 1% SDS) and resuspend the pellet with vigorous vortexing for 10 seconds.
11. Boil the samples for 5 minutes in a water bath then vortex them vigorously for 10 seconds.
12. Spin the samples for 15 minutes in a microfuge at room temperature.
13. Carefully remove 35 ul of the supernatant. Be sure and stay away from the bottom of the tube which contains invisible cellular debris that will contaminate the subsequent immunoprecipitations.
Note - It cannot be stressed enough that this is the most crucial step of immunopricipitation. For this reason 15 ul of the antigen extract is sacrificed so that one can stay away from the bottom of the tube and avoid contaminating material.
14. Add 1 - 4 ul of the appropriate antisera to 1 ml of ice cold triton buffer (50 mM Tris-Cl; pH 8.0, 0.15 M NaCl, 2% Triton X-100, 0.1 mM EDTA; pH 8.0). If required also add 50 ul of the appropriate cold competing antigen extract (see Appendex II for the preparation of cold competing antigen extract). Mix this solution very slowly and gently by inversion.
If the addition of cold competing antigen is required, let this mixture preabsorb on ice for 15 minutes.
15. Add the triton buffer, anitsera mixture (+/- cold competing antigen extract) to the 35S methionine antigen extract.
16. Mix very slowly and gently by inversion and place the resulting immunos on a rocking platform at 4oC for 3 hrs to 24 hrs.
17. Remove 20 ul of the immuno and place it in 10 mls of Safety Solve scintillation flour. Mix well and do a 2 minute count to quantitate the total cellular incorporation of 35S between different samples.
18. Add 50 ul of well-mixed swelled protein A-sepharose solution (0.1 g of protein A-sepharose is added to 2.8 mls of 10 mM sodium azide and allowed to swell o/n) to each immuno. Mix gently by inversion and place on a rocking platform at 4oC for 30 minutes to 1 hour.
19. Pellet the protein A-sepharose by microfuging at room temperature for 1 minute. Aspirate off the supernatant being careful to stay away from the protein A-sepharose pellet.
20. Do four 1 ml washes with ice cold triton buffer (vortex well). A 1 minute room temperature microfuge spin in sufficient to pellet the protein A-sepharose in between washes.
21. Do one 1 ml wash with ice cold 10 mM Tris-Cl; pH8.0, followed by one 1 ml wash with ice cold 0.1% SDS. After the last wash with 0.1% SDS try and remove as much of the supernatant as possible without removing any of the protein A-sepharose pellet.
22. Add 50 ul of SDS PAGE sample buffer to the protein A-sepharose pellet and vortex vigorously.
23. Boil the samples for 5 minutes in a water bath, then vortex them vigorously for 10 seconds.
24. Spin the samples for 5 minutes in a microfuge at room temperature.
25. Remove as much of the supernatant as possible ( 45 ul) and place in a new microfuge tube.
26. Load the samples after adjusting for differing amount of 35S incorporation. If the SDS PAGE gel is not to be run immediately, the immuno samples should be stored at -20oC. Frozen SDS PAGE samples must be thawed and reboiled for 5 minutes before they are run.
E. coli Immunoprecipitation
10X Low Sulphur Amino Acid Mix
600 mg L-Ala
500 mg L-Arg
700 mg L-Asp
800 mg L-Glu
300 mg L-Gly
200 mg L-His
300 mg L-Ile
1,100 mg L-Leu
700 mg L-Lys-HCl
300 mg L-Phe
300 mg L-Pro
300 mg L-Ser
300 mg L-Thr
400 mg L-Trp
200 mg L-Tyr
300 mg L-Val
qs to 1000mls with sterile ddH2O.
Filter sterilize and store in 100 ml aliquots at room temperature in bottles covered with aluminum foil to prevent the breakdown of tryptophan and tyrosine.
E. coli Immunoprecipitation
Preparation of Cold Competing Antigen Extract
If cold competing antigen is used in an immunoprecipitation it should be used at 10X the concentration of the 35S antigen.
1. Grow 2000 mls of the appropriate E. coli strain to an A600 of ~0.5.
2. Pellet and wash the cells with 250mls of 10 mM Tris-Cl; pH 7.5, 0.1 mM EDTA; pH 8.0.
3. Resuspend the cells in 8.0 mls of ice cold 50 mM Tris-Cl; pH 7.5, 10 mM EDTA; pH 8.0.
4. Add lysozyme, DNAse, and RNAse to 10 ug/ml (make up 1 mg/ml stock solutions) and PMSF (2 mg/ml in ethanol) to 20 ug/ml.
5. Freeze thaw three times in dry ice ethanol.
6. Add 1/10 volume (0.8 mls) of 1 M MgCl2, mix well by inversion, and incubate at room temperature for 30 minutes.
7. Add Triton X-100 to a final concentration of 2% (0.975 mls of a 20% Triton X-100 stock solution), mix well by inversion, and incubate at room temperature for 30 minutes.
8. Spin 15 minutes, 10,000 rpm, 4oC, and collect the supernatant.
9. Store the cold competing antigen extract in 0.5 ml aliquots at -70oC. The concentration of the cold competing antigen extract is 100 A600 units/ml.
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