Vawter Lab

Protocol available for download as .doc file HERE

Time Needed:
10-20 minutes per sample + 1 hr. setup/cleanup + 1/2 hr. per electrode cleanings
(Ex: 20 samples with 1 cleaning = ~ 6.5 hours)

Equipment Needed:
1) Beckman 350 pH/temp/mV meter (# 511211)
2) Beckman microelectrode (# 511082)
3) Beckman Saturated KCL for electrode (# 566468)
4) Fisher Calibration Buffers at pH 4, 7 & 10 (# SB101-500, SB107-500, SB115-500)
5) Reagents for cleaning electrode:

• a. 1 M HCL
• b. 1 M NaOH
• c. 10% bleach solution
• d. Acetone and Isopropyl alcohol
• e. 3-4 M Ammonium Hydroxide

6) Barnstead NANOpureInfinity ultrapure water system
7) Mettler Toledo AB54-S Analytical Balance
8) Centrifuge capable of 4C 5000 RPM for 2 minutes
9) -80C Freezer
10) Biospec Products Mini-Beadbeater-8
11) Biospec 1.0 mm glass beads # 11079110
12) Ice bucket and ice
13) Styrofoam box and dry ice
14) Aluminum foil
15) Standard straight edge disposable razors
16) Standard tweezers/forceps
17) Flat piece of glass (like the side of western gel apparatus)
18) Kimwipes
19) Tubes compatible with homogenizer & centrifuge (Sarstedt # 72.693.005), Standard 2.0 ml microcentrifuge tubes with the conical base (Fisher # 0566932)

Note:
a. There should not be more than an hour between taking a sample out in raw form and putting it back into the freezer after the pH has been read (this can usually be done by limiting the number of samples homogenized simultaneously to 4)
b. Do not use other beads - the zirconia beads have an average decreased pH of 0.39
c. Do not homogenize samples for more than 90 seconds with a Bead-Beater at max
d. If samples go beyond the recommended limits then those samples should be reprocessed and pH read again

Bead Prep:
1) Rinse the 1.0 mm glass beads in Nanopure water thoroughly
2) Transfer them to an autoclavable container and autoclave for 15 minutes
3) Dry the beads on a tray overnight in sterile hood
4) Transfer the beads back into the original bottle and cut off the tip so that the beads pour at a modest rate

pH Electrode Calibration:
1) Calibrate pH meter with fresh standards in 1.7 microcentrifuge tubes:
2) Clear off any loose KCl on the bottle or probe
3) Open the hole at the top of the probe by twisting gasket down
4) Unscrew the storage solution cap and position the probe
5) Make sure the KCL fluid is full in the probe and add if necessary
6) Turn machine on and rinse electrode with Nanopure water then blot with a Kimwipe
7) Put electrode into the tube and press Read
8) Wait for the eye to stop flashing and for the machine to zero in on the pH
9) If the reading is not within .02 of the calibration pH then the machine needs to be recalibrated
10) Press Cal button, then Press Clear button twice
11) Rinse electrode and place into pH 7 buffer and press Read
12) Repeat with second and third buffers (either 4 or 10)
13) Repeat for the pH 7 buffer a second time (to ensure accuracy)
14) Press Exit - the machine is now calibrated
15) Place the electrode into the pH 7 buffer and press Read
16) If the reading is not within .02 again then skip to 8f) then repeat 9), if this still doesn't do it then go to 10), if the reading is correct then place the electrode in the pH 4 buffer and continue to 12)

pH Electrode Cleaning:
1) Clean the bulb if necessary (30 minutes):
2) Soak electrode in 1M HCl for 5 minutes then rinse thoroughly with Nanopure water
3) Prepare another tube with 1M NaOH and let soak for 5 minutes, then rinse
4) For protein - soak electrode in 10% bleach for 5 minutes, then rinse
5) For surfactants - prepare a bottle with 200 ml acetone and 200 ml isopropyl alcohol
6) Dip the electrode into this solution briefly, then rinse with Nanopure water thoroughly
7) Let soak in pH 4 buffer for 5 minutes, then rinse and go onto step 9)
8) The problem may be the electrode junction - to clean that:
9) Soak electrode in 3-4 M Ammonium Hydroxide (NH4OH) for 25 minutes
10) Rinse with Nanopure and soak in pH 4 buffer for 15 minutes
11) Repeat 9) and if this still doesn't solve the problem then continue to 11)
12) Periodically salt buildup within the probe may cause problems - to clean:
13) Detach the electrode from the cable
14) Uncover the hole at the top and try to wrangle chunks of salt to the hole and let it fall out of the electrode
15) Heat some water to around 40C and immerse the entire electrode in the warm water
16) Try and dissolve salt build-up inside the electrode
17) Once all salt is dissolved let it set until it reaches room temperature
18) Empty electrode and rinse thoroughly
19) Replace the solution inside the electrode with fresh KCl
20) Repeat 9) - if this doesn't clear things up get a new electrode (discontinue protocol), if it does continue to 12)

Protocol:

Prep:
1) Add ~ 200 microliter beads to labeled tubes, the total volume of the beads should not exceed a tenth of the tube (or more than the ridge of the conical bottom)
2) Label tubes for homogenization and place them on ice with some Nanopure water
3) Turn on centrifuge and let it cool to 4C
4) Prepare dry ice platform:

• a. Get a Styrofoam box and place some flat pieces of dry ice into it
• b. Cover the entire surface with aluminum foil to contain tissue while cutting
• c. Place glass on the foil platform as a cutting surface

5) Clean tweezers and a disposable razor blade and let cool on glass
6) Scrape the frost off of the glass platform before handling the tissue, do this before each brain piece

Weighing and Homogenization:
7) From the -80C freezer retrieve the brain and place on platform
8) Break off a piece of tissue with razor and tweezers
9) Place a tube into the foam holder and weigh 50-90 mg of tissue
10) Calculate the amount of water needed for a 10% solution (mg x 10 = ul) for each sample and aliquot the appropriate amount of Nanopure water to each tube
11) Using a bead-beater, beat the pH samples for 60-90 seconds in the cold room - reprocess sample if homogenized for more than 90 seconds
12) Spin the pH tubes at 5000 rpm for 2 minutes at 4C

Measurement:
13) Rinse the probe with Nanopure water into the waste beaker
14) Warm the pH tube in your hands before putting it on the foam holder
15) Place pH probe into the homogenate until the bottom of the white area is covered (bulb fully covered)
16) Press Read and wait for the eye to stop blinking before recording the number
17) Rinse probe with Nanopure water and blot, then repeat for other samples
18) After every five samples or if a reading is > 7.2 or < 6.2 reread the calibration buffer 7 to ensure that the probe did not get clogged
19) If the reading is off, then let the electrode soak in pH 4 buffer for a couple of minutes and reread the pH 7 buffer (a fresh pH 7 tube)
20) If the reading is still off then the electrode requires cleaning - the tubes that have been prepared but not yet read should be placed immediately into -80C if there will be a delay
21) Place the electrode in pH 4 buffer between groups

Cleanup:
22) Save all tubes in -80C for rereading later
23) Rinse the probe and replace it back in the storage buffer vial
24) Turn off machine
25) When done with dry ice platform clean glass and tweezers, put dry ice and container away, wrap foil up with any tissue remaining and place in burn box, place razor in biohazard sharps container
26) Enter tissue amounts and pH into Excel