Lidstrom:in vitro Pathway Assay: Crude Extract Prep

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Crude Reaction Protocol

Do well in advance:

1. Outline goals (strains you will use, rxn conditions, # of biological & technical replicates)
2. Grow cells & store at -80oC
3. Chose a day to do extraction when you can work ALL DAY (& prepare the day before)
4. Reserve LCMS machine time

Do the night before:

1. Sign up for ultracentrifuge
2. Chill ultracentrifuge rotor in beer cooler
3. Decide how many cultures you will test (if not done yet)
   1. Can ultracentrifuge in sets of 4
      1. Each sample must be centrifuged (not ultracentrifuged) in 2 2 mL tubes because the rxn volume is close to 4 mL) but you can pool them again for ultracentrifugation
   2. Good to do an even number for centrifugation & ultracentrifugation
4. Enter data into excel sheet: determine how much of each reagent is needed
5. Make sure french press cells are clean & in the cold room
   1. May want to e-mail the group if you need exclusive access to the french press and will be using it at a peak use time. Also make sure there are enough parts, especially the white plastic balls and the donut & cylinder shaped o-rings. Always use one donut-shaped & one cylindrical o-ring.
   2. Fill the water & 70% EtOH bottles (for cleaning)
6. Weigh out approptriate amounts of each reagent (NAD+, NADH, ATP, etc.)
   1. some things you don't prepare: formaldehyde is in Amanda's fridge, 13C formate and pivolate are in Amanda's green bottom/white topped 20oC box, ThDP made by Amanda is stored in J's box and has green writing.
7. Prepare collection tubes
   1. don't need to autoclave.
8. Make phospate buffer, (currently pH 7 - 7.5 8/31/2012)
   1. 10X is for the reactions you set up; you add a lot of ATP, NADH, etc that doesn't have buffer in it, so you are making up for it by adding 10x.
      1. amount determined by the spreadsheet containing the reagent recipes
   2. PMSF 1X is for resupsending the cell pellet: this compound can help stabilize the proteins
      1. You will use 2.5 mL of 1X buffer with PMSF per sample (not currently using it?? 8/30/2012)
   3. 1X without PMSF is for washing the desalting columns. Make a lot! 40+ mL/sample you will desalt.
   4. Decide which solution you will use to do BCA BSA protein quantification and make a few mL extra for this (need ~ 1/10 mL per well plus some more)
9. Weigh out a suitable amount of BSA (2 mg/mL) for the BCA assay
10. prepare acetonitrile + pivolate: use spreadsheet to calculate the amount.

Morning of:

1. Chill ultracentrifuge rotor in beer cooler if you didn't the night before
2. defrost frozen tube cultures (see "cell pellet preparation") on ice
3. Resuspend the reagents (ATP, NADH, etc.) in the appropriate amount of water
4. cool centrifuge to 4oC, put in the rotor that holds eppendorf tubes, and put a sign up that you will be using it.

Crude Extract Preparation

1. Cool French press mini cell
2. Cool ultracentrifuge rotor
3. Defrost cell pellets on ice
4. Prepare protein buffer (5X) & 1X desalting/equilibration buffer
5. Resuspend cell pellets in 2.5 mL protein buffer (WAS 1x HEPES buffer + PMSF)

French Press

1. metal cell(s) should be cold (4^oC)
2. use small cell
   1. small cell sits on top of a metal cylinder with short pegs that hold it in place
3. insert fresh white bead/ball & check/replace seals on piston & bottom plug
4. lube pison @ seal end, and the plug
5. BEFORE LOADING SAMPLE:
   1. bottom plug in, exit nozzle attached (doesn't have to be super tight: after pressure drop), hand-crank valve tight
6. swing the arm that holds the cell in place into position
7. pressure = 1,000 PSI (maximum for the small cell)
8. ratio selector = "med" --> pressurizes cell
9. hold tube to nozzel as it lifts to reduce risk of it squirting out (if you don't have the crank tight)
10. unscrew so that ~ 1 drop/second enters tube.
11. keep tubes on ice between repeats
12. mark top of tube each time you french press it
13. send sample through a 2nd time
14. clean each cell before switching to another sample
15. alternate between EV & full construct to reduce bias of one cell pulverizing better or something
16. Move to *2 ml* centrifuge tubes if centrifuging (2 tubes/sample)
    1. If going straight to ultracentrifugation, add whole extract to ultracentrifuge tubes
17. Wash cell & parts and put cells back in 4^oC room

Centrifuge (optional)

1. Do this only if you aren't going to ultracentrifuge samples
2. Centrifuge 14,000 rpm , 4^o C for 20 min
3. Collect supernatant. This is the crude cell extract
4. Turn on ultracentrifuge and start cooling to 4^o C
5. Wash cell & parts if not done yet

Ultracentrifuge

1. Move CE to (disposable) polyallomer ultracentrifuge tubes
2. Use red anodized tube holder & mark what each is with sharpie & tape.
3. Balance tubes to nearest mg. 1 and 3 should match, 2 and 4 should match (for balancing)
   1. Use green test tube cap to hold it up.
   2. add ice cold buffer to samples you are diluting (not H₂2O)
4. Load "mushroom" into machine. Yes, it is surprisingly wobbly when you set it down, but that is really all you do.
5. Ultracentrifuge 45,000 rpm, 1 hr, 4^o C. (This will take longer than an hour due to spin up and spin down times)
   1. Takes ~ 1 hr 20 min with acceleration & deceleration
   2. Light blinks green after you start it: temperature needs to drop before it spins, even though everything is precooled
6. Collect supernatant in 2 fresh 2 mL tubes (per sample)
7. Collect up to 2.5 mL if desalting.
   1. This is the maximum (and minimum) volume of the desalting column
   2. Adjust volume to 2.5 mL with equilibration buffer
8. Wash cell & parts if not done yet

Desalting

(manual)

1. Desalting columns are stored with water filling the top, at 4^oC.
   1. New ones are stored in Ceci's fridge. Old ones are stored in Janet/Amanda's fridges. They are about $200-300/20 columns so we re-use them.
2. mark tube with date and # of uses
3. Put caps somewhere that they won't get lost
4. Desalting columns should be attended to so they don't dry out on top!
   1. Keep a close eye out so the top surface essentially never runs out of water.
5. Equilibrate PD-10 desalting column and desalt HSFs two times. Don’t need to wash column as much between 1st and 2nd passes through the column.
   1. Wash ~ 7X with buffer before loading first sample.
   2. Wash with buffer 3 times between re-running the same sample (don't count the volume used to elute your sample)
      1. When loading samples, wait ~ one second after the water on top of the stationary phase disappears.
      2. Load 2.5 mL, not more or less.
      3. Once all of the solution enters the column, elute by adding 2.5 mL of buffer
         1. Collect in the ~ 12 ml plastic falcon tubes with clear lids. You can tape it to the box to collect the eluate
      4. Collect 2.5 mL (or less if you are on your last sample and don't need it all)
   3. Wash ~ 7X again after all samples have been run.
6. At end, wash column with ddH2o water (7X) and store with ddh2o water. (both ends capped)
   1. Rinsing out buffer before storage.

Reactions

1. Make reaction master mixes (1 tube per row on excel sheet)
2. Setup reaction tubes (# of rows * number of samples in each row)
3. Start reactions by combining master mix with protein solution
   1. Quench 50 uL of reaction into 150 uL of acetonitrile + pivolate for each timepoint
   2. Do time = 0 quickly
4. Store quenched samples at -20oC.
5. acetonitrile + pivolate doesn't have to be refrigerated (if you distribute it into the collection tubes) but it doesn't hurt

BCA (protein concentration determination):

1. Clean 96-well plates to load samples in are in the right cabinet above the GC
   1. Use the flat-bottomed ones. (? what are the round bottom ones for?)
2. Plan out what samples you want to run & where they will go on the 96 well plate (find your old plate)
   1. If doing more than one set of standards, put one set at the beginning and one at the end. Betsy says the end set is always slightly less developed. Check up on this. Use the average, I guess.
3. Make protein standards. Make 1/3 to 1/4 of the volume recommended by scaling back the volumes stated by a factor of 3 or 4.
4. Prepare protein dilutions. Do 1/5 conc after desalting, 1/10th conc after desalting, and 1/25th.
   1. Prepare these in 100 uL in PCR tubes to allow for use of the multichannel (parallel) pipette.
5. load samples into well
6. prepare working reagent (WR)
7. use multi-channel pipette to distribute the WR into the wells
8. incubate at 37^oC for 30 min
   1. Ceci says < 30 min is ok, but suggested > 30 min is not good. Janet: look this up.
9. measure with the plate reader at Betsy's bay.

Also need to

1. Nash reagent?
2. BCA assay
   1. can I freeze aliquots to do the next day?

At the end of the day:

1. go around to each piece of equipment & ensure it is cleaned up
   1. french press area cleaned, centrifuges turned off