Team:Calgary/Notebook/Protocols/Process22

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Biotinylation and (Theoretical) Immunoprecipitation of Cyclohexanepentanoic Acid

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:

  • Add10 uL of pure cyclohexanepentanoic acid 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 hydroscopic and must be kept away from moisture. Store the solution you make at -20C in the biotin box!
  • Make up a stock solution of 100 mM Biotin by weighing it out into a 1.5 mL Epindorf 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 -20C 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 (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 uL of pre-washed streptavidin coated magnetic beads (SoluLink) 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. 10. Allow the solution to go EOE for 2-5 min. and repeat this procedure 2 more times. (i.e. force the beads to the side of the tube, remove the supernatant, and add more lysis buffer). YOU SHOULD AT THIS POINT HAVE 8 TUBES, 4 with only BIOTIN, and another 4 which had your reaction in it that have been cleaned using lysis buffer. IF YOU SEE ANY STRANGE PRECIPITATION OR CHANGES IN COLOUR, or ANOTHER SOLVENT LAYER please let me know. WRITE IT DOWN! 11. Remove the lysis buffer supernatant after moving the beads to the side of the tube and add pseudomonas lysate to the tubes. (see below for more details). The protocol below is for making your pseudomonas lysates: 1. You are making two kinds of lysates. One that has been crosslinked for chIP (determine the DNA content) and another which has not for regular IP (for protein identification) 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) follow protocol 1. 2. For the crosslinked chIP’s follow protocol 1 as you have before, including the sonications that you have done previously for our successful shearing (you should have 1 putida and 1 flourescens for the crosslinked and 1 of each for the non-crosslinked). Protocol 1 has been altered slightly because we want to make a large amount of lysate so please read the next page carefully. Protocol 1: 1. Add 27μl formaldehyde (37%) per ml medium (subtract what you took out for measuring OD) => final concentration of about 1% to the crosslinked samples you have grown up. DO NOT ADD FORMALDEHYDE to the REGULAR IP TUBES. Follow steps 2-4 for only the crosslinked samples just keep the regular IP cultures growing. 2. Shake slowly (100 RPM) for 20 min at RT (feel free to modify these steps if you have done something different that I am not aware of when you were doing the shearing). 3. Add 10 ml of 2.5 M glycine => final concentration of about 0.5 M 4. Keep shaking for 5 min 5. Harvest all of the solution in 10 ml volumes of cells for each preparation (centrifuge 2500 g, 4°C, 10 min) (i.e. you will have 5 tubes for each culture you set up) DO STEPS 5-11 for all tubes 6. For each tube, wash twice in cold 10 ml TBS (20mM; see Material) pH7.5 (You can freeze the cell pellet and proceed later (better to freeze than not)) 7. Resuspend in 1 ml Lysis-Buffer 8. Incubate at 30 °C for 30 min (not shaking) 9. Add 4 ml of IP-Buffer 10. Add 50 μl of 100mM PMSF => final concentration of 1mM 11. Sonicate so that DNA fragments are <1 kb on average SONICATION TIMES: ______________ ________________ _________________ 12. For the regular IP samples: Pipette the sonicated solution into 1.5 mL Epindorf tubes and spin them down at 4 degrees (in the cold room if there is a centrifuge there) in a table top centrifuge at 14,000 rpm for 15 min. Once it is finished collect the supernatant and pool it into 15 mL Falcon tubes. Leave out _______ mL of lysate and freeze the rest immediately in the -80C Freezer. LABELLING HINT! Make sure that you have the Date, Initials, Name of the Lysate and what it exactly is on the tubes. You will have to go back to do this, but also write the protein concentration of the lysate which you will do by Bradford Assay in a few steps. Also parafilm the tops For the crosslinked IP samples: Centrifuge at 12,000 g 4C for 10 min. FROM THIS STEP ONWARDS IT IS CRITICAL THAT EVERYTHING REMAIN ON ICE AT ALL TIMES. IF NOT THEN YOUR PROTEINS AND DNA WILL GET DEGRADED. From this point on the protocols are going to become a little more separated and it may be hard to budget your time effectively. Hopefully by this point I can come over and help you guys but it should be pretty easy. 13. Perform a Bradford assay and determine the concentration of the lysates that you have made for regular IP. Make sure that you write it down. If you need to know how to do this ask Deirdre. She should have all the stuff for it. Don’t worry about doing this for the other lysates (that were crosslinked for chIP) just move on to the next step for them. Protocol: ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ ____________________________________________________________ 14. Add 1.3 mL ________ mg of regular non-crosslinked lysates (putida and flourescens) to two of the prepped magnetic beads (one which has the biotinylated reaction that you did yesterday, and the other one the control biotin beads – labeling is important here) that you have made and resuspend the beads gently with a pipette. To the other two tubes add 800 uL of the crosslinked samples to two of the tubes. 15. Add 8 uL of 100 mM PMSF (stored in the -20 Freezer in the iGEM lab) to the crosslinked tubes and 13 uL of PMSF to the regular IP tubes. Let them go EOE at 4C O/N in the cold room. Make sure the tops are well closed.


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