Biotinylation/Immunoprecipitation of NA's

Day 1

1. Inoculate Cultures in 50 mL Tubes (whatever cultures you are interested in probing) with and without the compound you are sensing.

2. At the same time set up a biotinylation reaction as follows, dissolving all solutions in 80% DMSO:

Add 10 uL of pure cyclohexanepentanoic acid (CHPA) to 167.10 uL of 80% DMSO
Make up a solution 500 mM EDAC in 80% DMSO. (Dissolve 10 mg of EDAC in 0.1 mL of 80% DMSO) Add 12.5 uL of the EDAC to the cyclohexanepentanoic acid solution you made. Don’t keep the EDAC out very long it is very hygroscopic and must be kept away from moisture. Store the solution you make at -20°C in the biotin box!
Make up a stock solution of 100 mM Biotin by weighing it out into a 1.5 mL Epindorff on an analytical balance (be careful when you do this) spin down the tube then add 80% DMSO as before. Again store this solution at -20°C in a tightly sealed container.
pH the solution using 5% HCl (found in the acid cabinet) to pH 4-7. Optimal pH of the solution appears to be between 5-5.5. Add 2 uL of the acid to the solution and mix well. Take a few microlitres of the solution and spot it onto a piece of pH paper. Repeat if necessary
Let the solution sit at room temperature O/N.

3. Set up a second reaction for control with biotin only in 80% DMSO.

Add 12.5 uL of the Biotin solution that you made into 187.5 uL of 80% DMSO. pH the solution to roughly the same pH as your reaction using 5% HCl (using ~2uL) and let it sit O/N like the other reaction at room temperature.

FINAL CONCENTRATION OF THE SOLUTION 10 mg of cyclohexane pentanoic acid (CHPA) (or 54mM) 5 mM EDAC 5 mM Biotin

Day 2

Continue the biotinylation from the previous day:
4. After the 24 hours, take 25 uL of the solutions and freeze them for HPLC analysis.
5. Take both biotinylation solutions and dilute the samples in 1.8 mL of PBS solution (50 mM Sodium Phosphate, 15 mM NaCl, pH 8.2), mix well.
6. Add 50-100 uL of pre-washed streptavidin coated magnetic beads (Invitrogen-270) to each solution and allow them to go end-over-end for 2 hours at room temperature.
7. After incubation, apply the magnet to the side of the tube and wait for the beads to collect (this should only take about 1 min. but make sure you get them all). Remove the solution using a pipet tip being very careful not to touch the beads. Wash the beads with 1 mL of PBS.
Prepare Lysates of Your Favorite Organism: (Following is the lysate protocol we used for producing Pseudomonas lysates adapted from OpenWetWare ("http://www.openwetware.org/wiki/ChIP-Chip_E._coli").

Once your cells have grown and reached an optical density OD₆₀₀ of at least 0.5 (O/N should have been good enough but you might want to check just in case), centrifuge 2500xg, 4°C, 10 min.
For each tube, wash twice in cold 10 ml TBS pH 7.5 (20mM Tris, 150 mM NaCl) (You can freeze the cell pellet and proceed later if required).
Resuspend in 1 ml Lysis-Buffer (10mM Tris pH 8.0, 20% sucrose, 50mM NaCl, 10mM EDTA, 10 mg/mL lysozyme)
Incubate at 30°C for 30 min (not shaking)
Add 4 ml of IP-Buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1mM EDTA, 1% Triton, 0.1% Sodium deoxycholate, 0.1% SDS)
Add PMSF to a final concentration of 1mM
Sonicate the solutions 9 times 30 sec. with 30 sec. of rest in between using a Branson 450 sonicator at an output of 15%.
Clarify the solution by centrifugation at 14,000 RPM for 15 min. at 4°C
Determine protein concentration by Bradford Assay.

8. Add your washed beads to a solution of ~5-6mg of lysate and incubate with PMSF (1mM final) for 2 hours end-over-end at 4°C.

9. Wash the beads with 4 rounds of 150mM NaCl PBS (1mL) for 5 min. each round.

10. Elute the solution in 30 µL SDS-PAGE Loading Buffer (1x) twice, boiling for 5 min. in between each round.

11. Run the solution on a gradient gel, identify protein interactors by sending for MS sequencing.

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Biotinylation/Immunoprecipitation of NA's

Day 1

Day 2