Protocols

Neuron Culture Solutions  |  Neuron Culturing  |  Transfecting Neurons with Lipofectamine 2000  |  Preparation of Brain Membrane Fractions  |  Biotinylation Assay of Receptor Endocytosis with Cleavable Biotin Reagent  |  PSD Prep from High-Density Cultured Neurons  |  Large-batch Procedure for Preparing PDL-coated Coverslips



Hanks Plus ( HBSS+)

• HBSS (w/o Ca2+, Mg2+)
• + 10mM HEPES
• + 33.3mM Glucose
• + 5ug/ml Gentamicin (1:10,000 dilution of 50mg.ml Gentamicin)

Dissection Medium

• Hanks Plus (HBSS+)
• + 0.3% BSA
• + 12mM MgSO4

Culture Medium

• Neurobasal Medium (100ml)
• + B27 supplement (2ml)
• + GlutaMAX I (1ml)
• + 1ug/ml Gentamicin (2ul of 50mg/ml)

Digestion Solution

• NaHCO3 (4.2mM)
• HEPES (25mM)
• NaCl (137mM)
• KCl (5mM)
• Na2HP4 (7mM)
• Adjust to pH 7.4

FUDR:

• 5-FLUORO-2?DEOXYURIDINE (F-0503 100mg SIGMA) 10mM 25mg
• URIDINE (U-3003 5g SIGMA) 10mM 24mg
• H2O
• Final Volume: 10ml
• Sterile filtration, 0.2um

AraC: Cytosine ?D-Arabino-Furanoside

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Coating (day before dissection)

Poly-D-lysine
1mg/ml 80ul/glass coverslip (or enough to cover the coverslip) 0.1mg/ml
2ml/60mm dish
Incubate at 37 or RT, O/N (or can go as short as 2 hrs if in a hurry)
(Day of dissection)
Wash coverslip or dish with water 2X


Laminin
1ug/ml 2ml/60mm dish (after dishes were coated with PDL)
Incubate at least 2h
Wash dishes with HBSS 2X.
Dish and plate can be filled with plating medium, keep in incubator


Dissection

Prepare ice-cold Dissection Medium (DM) w/CaCl2 (2mM) in dish and two 15ml-tube
All instruments in 70% Alcohol
4-6 Rats
Oval anesthesia: Halothane


1. Cut head right behind ears (large scissors and forceps with teeth) into dish cover


2. Cut skin and skull through the middle line, two cuts behind eyes (10cm scissors)

3. Lift skin and skull to expose brain (10cm graefe forceps)


4. Move brain into dish w/DM (Micro-spatula, a little bit curved)

5. ( 8cm angled-up spring scissors and 11cm fine shanks forceps)


6. Tear off the vessel membrane outside the cortex (11cm fine shanks forceps and 45 tip forceps)

7. Expose hippocampus (11cm fine shanks forceps and 45 tip forceps)

8. Cut hippocampus along outside line and roll it off ( 11cm fine shanks forceps and 8cm spring scissors)



9. Collect all the cortex to a dish w/DM, chop them into pieces and pipette them in 15ml tube w/DM.

10. Roll and open hippocampus and cut correct region of hippocampus (11cm fine shanks forceps and scalpel), collect and chop them into pieces, pipette them in 15ml tube w/DM


Digestion

1. Spin down for 1min. 1000rpm.

2. *Rinse with Hanks Plus 10ml/tube

3. *Spin down for 1min. 1000rpm.

4. Dissolve 70mg Trypsin and 2mg DNAse in 8ml pre-warmed (37 ) Digestion Solution. Filter sterilize the digestion solution (DS).

5. Aspirate the Hanks Plus and incubate cortex pieces in 5ml sterile DS and hippocampus pieces in 3ml sterile DS, 5-8min. 37 agitate slowly from time to time.

6. Spin down for 1min. 1000rpm. And aspirate the supernatant.

7. During incubation, dissolve 40mg trypsin inhibitor in 8ml pre-warmed (37 ) Dissection Medium (DM). Filter sterilize to new tube.

8. Incubate hippocampus pieces with 3ml DM with trypsin inhibitor (5ml for cortical pieces) for 2min at 37 .

9. Spin down for 1min. 1000rpm. And aspirate the supernatant.

10. Wash with 10ml/tube ice cold DM. Spin down for 1min. 1000rpm. And aspirate the supernatant.

11. Dissolve 1mg DNAse in 5ml ice cold DM, filter sterilize. Add 1ml to hippocampus pieces and 3-4ml to cortex pieces.

12. Triturate slices very gentle. Avoid air bubbles (osmotic shock). First, use a fire polished glass Pasteur pipette, then use a fire polished pipette with reduced diameter

13. *Filter cells through the cell strainer (40um Falcon 2340), rinse with some DM.


Cell recovery

1. Spin down for 10min. 1000rpm. And aspirate the supernatant.

2. Re-suspend the hippocampus pellet in 5ml ice-cold DM or Hank's Plus and the cortex pellet in 5-10ml ice-cold DM or Hank's Plus.

Counting

10ul cells + 90ul Trypan Blue Stain 0.4% (15250-061 GIBCO)

Pipette 10ul of mixture above on Counting Chamber (15170-208 VWR)

The cell number inside 4x4 square will be N,

N x 105cell/ml --------cell concentration


Plating

Plate 2 million (106) cortical cells on each dish with Plating Medium.

Plate high or low density of hippocampal cell on each coverslip with Plating Medium.


Feeding

(3 days after culturing) Add FUDR (10uM) to cortical neurons.

(wait for perfect time - Glial cell layer reaches around 70% confluence) Add FUDR (10uM) to hippocampal cells

(twice a week) Replace half of the medium with fresh Culture Medium w/FUDR (10uM) for hippocampal neurons.

(twice a week) Replace half of the medium with fresh Culture Medium for cortical cells.

*: Optional steps that can be skipped. We usually skip them.

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To transfect cells in one well of a 12-well plate (~1ml growth medium in the well):

1) Mix 25µl Opti-MEM with 1µl Lipofectamine 2000. Let sit for 5 min at room temperature.

2) Mix 25µl Opti-MEM with 1 to 1.5µg cDNA of choice (for co-transfections, use 1 to 1.5µg of each construct). This does not need to sit. It is possible to use much less DNA.

3) Combine solutions from 1 and 2, and pipette gently to mix. Let sit for 20 min at room temperature.

4) Add total solution from 3 to well. Swirl to mix in the well.

Notes:

I never wash off the growth medium, except if I've transfected freshly plated neurons.

Using Opti-MEM is critical when pre-mixing the L2K. The neurons can be growing in Neurobasal and serum, though, when the mixture is added.

Transfection rates are quite high during the first week after plating (<10%) but very low after that. Expect only a few neurons per well to be transfected after a couple of weeks.

Dense cultures transfect much, much better than sparse ones, perhaps due to the presence of glia.

Expression starts to be visible in ~2-4 hours, and peaks after 24 hours.

Duration of expression is totally dependent on the construct.

Cell lines can be transfected efficiently with L2K as well, using 1/10th or less of the lipid.

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Crude Membrane/Cytosol Prep Be sure that all procedures are done with precooled reagents at 4°C.

Dissect out brain regions of interest into ice-cold into 10 volumes of cold homogenization buffer (0.32 M sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, protease inhibitors, phosphatase inhibitors as below).

Homogenize using 10-15 strokes of a motor-driven glass-teflon homogenizer. Never use polytron.

Spin at 1000 x g for 15 min to remove pelleted nuclear fraction (P1).

Take supernatant (S1) and spin at ~200,000 x g (50,000 rpm for 30 min in 70.1 Ti rotor; 62,000 rpm for 15 min in TLA100.3 rotor) to yield crude cytosol (S2) and crude membrane pellet (P2).

Resuspend pellet in homogenization buffer.

Spin again at ~200,000 x g to yield washed crude membrane pellet (P2?.

Resuspend pellet in HEPES-Lysis buffer (50 mM HEPES pH 7.4, 2 mM EDTA, protease/phosphatase inhibitors).

Measure protein concentration by BCA or Coomassie.

Can solubilize with detergents or store at ?0°C.


Microsome Prep

Microsomes = vesicles derived from rough ER and various smooth membrane bound organelles (including Golgi stack components).

Homogenize brain in homogenization buffer (0.32 M sucrose, 10 mM HEPES pH 7.4, 2 mM EDTA, protease inhibitors, phosphatase inhibitors as below).

Spin at 1000 x g for 15 min to remove pelleted nuclear fraction (P1).

Take supernatant (S1) and spin at 10,000 x g for 20 min to remove pelleted mitochondria (P2).

Spin supernatant again at 12,000 x g for 30 min to doubly remove pelleted mitochondria (P2).

Take resulting S2 supernatant and centrifuge at 140,000 x g for 120 min to yield microsomal pellet (P3).

The resulting microsome pellet can be resuspended in homogenization buffer and further fractionated by layering over a discontinuous sucrose gradient (0.8, 1.0, 1.3, and 2.0 M sucrose) and centrifuging at 97,000 x g for 120 min.

I can’t quite remember what organelles are present at each interface, but Wenthold typically uses the interfaces between 1.0 and 0.8 M and between 0.8 M and 0.32 M.

For reference, see Gurd et al., (1974). J. Neurochem. 22:281-290.


Synaptic Plasma Membrane Prep

This protocol is adapted from Blackstone et al. (1992) and Lau et al. (1996) and should be followed when an enriched membrane prep or PSD fraction is desired

All procedures should be done at 4°C using precooled reagents. For rat and mouse samples, immediately remove brain from the cranium into ice cold HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4) containing a freshly added protease inhibitor cocktail (required, see below) and phosphatase inhibitor cocktail (optional, see below). Different parts of the brain can be subdissected and enriched plasma membranes/PSD can be prepared as described below.

Protease Inhibitors	Phosphatase Inhibitors
PMSF (0.1 mM) Aprotinin (1.5 µg/ml ea.) Antipain/leupeptin (10 µg/ml ea.) Chymostatin/pepstatin (10 µg/ml ea.) Benzamidine (0.1 mg/ml)	EGTA (2mM) NaF (50mM) sodium pyrophosphate (10mM) ?glycerophosphate (20mM) para-nitrophenylphosphate (PNPP) (1 mM) microcystin LR (optional) (1 µM) Sodium orthovanadate (1 mM) Ammonium molybdate (0.1 mM)

1.Add 10 volumes of HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH7.4) to the tissue and homogenize in a motor driven glass-teflon homogenizer at ~900 rpm (10-15 strokes). Never use polytron.

2. Centrifuge the homogenate (Hom.) at 800-1000 x g at 4°C to remove the pelleted nuclear fraction (P1).

3. Spin resulting supernatant (S1) at 10,000 x g for 15 min (9200 rpm in SL50T rotor) to yield the crude synaptosomal pellet (P2).

4.Resuspend pellet (P2) in 10 volumes of HEPES-buffered sucrose and then respin at 10,000 x g for another 15 min to yield the washed crude synaptosomal fraction (P2?.

5. Lyse resulting pellet by hypoosmotic shock in 9 volumes ice cold H20 plus protease/phosphatase inhibitors and three strokes of a glass-teflon homogenizer

6. Rapidly adjust to 4 mM HEPES using 1 M HEPES, pH 7.4 stock solution.

7. Mix constantly in cold room for 30 min to ensure complete lysis.

8.Centrifuge lysate at 25,000 x g for 20 min (14,500 rpm in SL50T rotor) to yield a supernatant (S3, crude synaptic vesicle fraction) and a pellet (P3, lysed synaptosomal membrane fraction).

9. Resuspend P3 pellet in HEPES-buffered sucrose.

10. Using a Pasteur pipet, layer the resuspended membranes on top of a discontinuous gradient containing 0.8 to 1.0 to 1.2 M sucrose (top to bottom; equals 27%/34%/41%) in a clear tube. Don’t forget to add protease/phosphatase inhibitors to the sucrose solutions! Always use a Pasteur pipet to pour layers and add sample.

11. Centrifuge the gradient at ~150,000 x g for 2 hr in a swinging bucket rotor (30,000 rpm in SW41 Ti; 36,000 rpm in SW50.1 Ti; 42,000 rpm in TLS-55; 28,0000 rpm in SW28).

12. Recover synaptic plasma membranes in the layer between 1.0 and 1.2 M sucrose.

13. Dilute to 0.32 M sucrose by adding 2.5 volumes of 4 mM HEPES pH 7.4.

14.Pellet by centrifugation at 150,000 x g for 30 min. 42,000 rpm in 70.1 Ti; 55,000 rpm in TLA 100.3

15.Resuspend resulting pellet (SPM) in PBS (pH 7.4) or (50 mM HEPES pH 7.4, 2 mM EDTA) with protease and phosphatase inhibitors.

16. Can store at ?0°C.



Postsynaptic Densities

For references, see Carlin et al. (1980) and Cho et al. (1992).

1. Resuspend synaptic plasma membranes prepared as above in 3-5 ml of ice cold 50 mM HEPES pH7.4, 2 mM EDTA, plus protease/phosphatase inhibitors. Add Triton X-100 to 0.5%

2. Rotate in cold room for 15 min.

3. Centrifuge at at 32,000 x g for 20 min (22,000 rpm in 70.1 Ti; 28,000 rpm in TLA 100.3) to obtain the PSD-1T pellet.

4. Resuspend PSD-1T in 3 mL ice-cold 50 mM HEPES pH7.4, 2 mM EDTA plus protease/phosphatase inhibitors). Save 1 mL.

5. To half of the remaining resuspended pellet (~1 mL), add Triton X-100 to 0.5% and rotate in cold room again for 15 min.

6. Centrifuge at 200,000 x g for 20 min to obtain the PSD-2T pellet. 50,000 rpm in 70.1 Ti; 65,000 rpm in TLA 100.3.

7. In a separate experiment resuspend the second half of the PSD-1T pellet and incubate for 10 min in ice-cold 3% sarcosyl (same as N-lauroyl sarcosine) in 50 mM HEPES pH7.4, 2 mM EDTA, plus protease/phosphatase inhibitors.

8. Centrifuge at 200,000 x g for 1 hr to obtain the PSD-1T+S pellet.

9. All pellets can be resuspended in PBS or 50 mM HEPES pH7.4, 2 mM EDTA plus protease/phosphatase inhibitors or SDS-PAGE sample buffer.

10. For PSD-2T pellets, may need some SDS (0.2%) to dissolve completely.


Synaptic Vesicle Prep

1. To prepare synaptic vesicles, prepare S3 fraction as in Synaptic Plasma Membrane Prep above.

2. Centrifuge S3 fraction at 165,000 x g for 2 hr. 55,000 rpm in TLA 100.3, 45,000 rpm in 70.1 Ti.

3. Resuspend in PBS or 50 mM HEPES pH7.4, 2 mM EDTA plus protease/phosphatase inhibitors.


Pure Synaptosome Prep

For crude synaptosomes, can simply use P2?fraction from the Synaptic Plasma Membrane Prep.

For added purity:

1. layer P2?fraction onto 4 ml of 1.2 M sucrose.

2. Centrifuge at 230,000 x g for 15 min in swinging bucket rotor. 38,000 rpm in SW 41 Ti, 44,000 rpm in SW50.1, 52,000 rpm in TLS-55.

3. Collect gradient interphase.

4. Dilute to ~7-8 ml with ice-cold HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH 7.4).

5. Layer onto 4 ml of 0.8 M sucrose.

6. Centrifuge at 230,000 x g for 15 min.

7. Pellet contains pure synaptosomes.

8. For bioassays, wash and resuspend in appropriate incubation buffer:

For electroporation or functional release assays: (140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1 mM MgCl2, 20 mM HEPES pH 7.4, 10 mM dextrose (added fresh), ?.8 mM CaCl2, and optional ATP regenerating system.

For digitonin permeabilization: 115 mM KOAc, 25 mM HEPES, pH 7.4, 5 mM NaOAc, 0.05 mM EGTA containing 0.025% digitonin, an ATP regenerating system, and 10 mM DTT. (see Meacham, Patterson, et al., Nature Cell Biology, 2001)

ATP regenerating system ?5 mM Mg-ATP, 80 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase

9. Otherwise, can solubilize as desired, or subfractionate further into synaptosomal membranes and synaptic vescles by hypotonic lysis as described under Synaptic Plasma Membrane Prep.


Synaptoneurosomes

Advance Prep

a. Get rat

b. Prepare Dissection Buffer (~100 mL, see below) and oxygenate on ice.

c. Prepare Incubation Buffer (see below) and place on ice and 37°C if needed. Need ~30 mL/brain.

d. Put glass-glass Dounce homogenizer and 2-3 conical tubes on ice. (1 brain requires 15 mL Dounce homogenizer).

e. Prepare nylon syringe filters (100 m and 50 m). To do this, cut ends off of 30 mL syringes, warp double layer of nylon mesh over end and fasten tightly with rubber and (thick rubber bands for biohazard waste work well for this).

f. Prepare Millipore Mitex 10 m filter (either the #LCWP 047 or LCWP 025) by fitting to Swinnex filter holders (Millipore). Be sure to tighten these HARD.

g. Get dissection tools/guillotine

h. Set up protein assay

1. Rapidly dissect out brain regions of interest in ice-cold oxygenated dissection buffer (see below) (212.7 mM sucrose, 2.6 mM KCl, 1.23 mM NaH2PO4, 26 mM NaHCO3, 10 mM dextrose, 10 mM MgCl2, 0.5 mM CaCl2, saturated with 95% O2 and 5% CO2). One can optionally include 20 mM CNQX plus 100 mM AP5 if excitotoxicity is a problem. Can also substitute kynurenic acid (2 mM) for CNQX and AP5.

2. Homogenize in 10 volumes ice-cold incubation buffer: 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1 mM MgCl2, 20 mM HEPES pH 7.4, 10 mM dextrose (added fresh), ?.8 mM CaCl2, and optional ATP regenerating system by 7 strokes with a loose pestle and 4 strokes with a tight pestle in a glass-glass tissue homogenizer (Kontes). Add protease inhibitors and phosphatase inhibitors if only using synaptoneurosomes for western. If using SN’s for bioassay, leave out protease inhibitors and phosphatase inhibitors unless testing pharmacologically. Don’t forget to add ubiquitin aldehyde for ubiquitination experiments!!!

3. SAVE ALIQUOT OF UNFILTERED BRAIN HOMOGENATE.

4. Pass homogenate over two layers of 100 mm pore nylon mesh filters (Small Parts, Inc.) pre-wetted with cold incubation buffer followed by two layers of pre-wetted 50 mm pore nylon mesh filters (optional) (Small Parts, Inc). This step is accomplished by cutting the ends of 20 mL syringes and fastening the nylon mesh tightly over using rubber bands. Homogenate is then placed in the syringe and gentle pressure applied.

5. Force homogenate through a final pass pre-wetted 10 mm pore LCWP 047 or LCWP 025 Millipore Mitex membrane filter (Millipore catalog # LCWP 047 00 and LCWP 025 00) fitted to Swinnex filter holders (Millipore). Make sure filter holder is VERY tight.

6. Collect the filtered particulate and spin at 1000 x g for 20 min at 4°C.

7. Discard supernatant.

8. Resuspend pellet in 5 volumes incubation buffer.

9. Respin at 1000 x g for 10 min at 4°C to obtain washed synaptoneurosomes.

10. Resuspend pellet in incubation buffer (~ 5 volumes) to a final protein concentration of ~ 0.5 ?1.5 mg/ml. Yield is approximately 20 mg SN/brain

11. Use synaptoneurosomes fresh for experiments (within 90 min) or freeze at ?0°C if only to be used for western blot.

12. For bioassay experiments, may also want to add an ATP regenerating system, as well as some DTT (10 mM) if the synaptoneurosomes will be permeabilized or electroporated. See above section of synaptosomes for electroporation and digitonin solutions.

ATP regenerating system ?5 mM Mg-ATP, 80 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase

Solutions
Stock Dissection Buffer	1L
KCl (2.6 mM)
NaH2PO4 (1.23 mM)
NaHCO3 (26 mM)
Kynurenic Acid (2 mM)
Dissection Buffer	100 mL	made fresh
Stock Dissection Buffer	100 mL
Sucrose (212.7 mM)	7.28 g
Dextrose (10 mM)	0.18 g
1M MgCl2 (10 mM)	1 mL
1M CaCl2 (0.5 mM)	0.05 mL
Stock Incubation Buffer	1L
NaCl (140 mM)
KCl (5 mM)
NaHCO3 (5 mM)
Na2HPO4 (1.2 mM)
HEPES pH 7.4 (20 mM)
Incubation Buffer	50 mL	100mL	made fresh
Stock Incubation Buffer	50 mL	100 mL
1M MgCl2 (1 mM)	0.05 mL	0.1 mL
1M CaCl2 (1.8 mM)	0.09 mL	0.18 mL
Dextrose (10 mM)	0.09 g	0.18 g

Rotor Notes

Available rotors at Duke:

For our SuperT21 table top superspeed:

ST-H750 (swinging bucket, ~700 ml, max 3800 x g), SLC-250T (fixed angle, ~200 ml, max 20,000 x g), SL-50T (fixed angle, ~25 ml, max 41,000 x g).

For the floor model Beckman XL-90 ultracentrifuge

Dept. of Neurobiology

Type 70.1 Ti (fixed angle, max 450,000 x g), SW28 (swinging bucket, max 140,000 x g, 6 x 39 mL), NVT90 (near vertical, max 645,000 x g)

Dept. of Neurology

SW41 (swinging bucket, max 288,000 x g, 6 x 13.2 mL), SW 50.1 (swinging bucket, max 300,000 x g, 6 x 5 mL), Type 60 Ti (fixed angle, max 360,000 x g)

For the Beckman tabletop ultracentrifuge in Pate’s lab

TLA 100.3 (fixed angle, max 540,000 x g), TLS-55 (swinging bucket, max 255,000 x g)

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Add 100 micrograms/ml of the lysosomal protease inhibitor leupeptin 30 min prior to biotinylation.

Rinse cells gently with progressively cooler PBS++ (phosphate-buffered saline plus 1 mM MgCl2 and 2.5 mM CaCl2) (RT, 10C, 4C)

Incubate with 1.5 mg/ml sulfo-NHS-SS-biotin in PBS++ (phosphate-buffered saline plus 1 mM MgCl2 and 2.5 mM CaCl2) for 20 min at 4°C. Make this up fresh.

This cell-impermeable reagent covalently conjugates biotin to primary amine groups of proteins, coupling the biotin to proteins via a reversible disulfide linkage.

Quench unreacted biotin with cold 50 mM glycine in PBS++ (rinse once, then 2 x 5min).

For determining surface expression, proceed directely to cell lysis/membrane prep.

For endocytosis assay, incubate cells at either 4°C to block membrane trafficking (negative control) or 37°C for various times to allow endocytosis to occur.

Cleave remaining surface biotin by reducing the disulfide linkages with glutathione cleavage buffer (50 mM glutathione in 75 mM NaCl, 10 mM EDTA containing 1% BSA, 0.075 N NaOH) (2 x 15 min, 4ºC).

Quench unreacted glutathione with 5 mg/ml iodoacetamide in PBS++ (rince once, then 2 x 5 min) before cell lysis.

Membrane Prep

Scrape cells into cold lysis buffer [50 mM Tris pH 7.4, 2 mM EDTA, 2 mM EGTA, plus phosphatase inhibitors (50 mM NaF, 10 mM sodium pyrophosphate, 20 mM b-glycerophosphate, 1 mM p-nitrophenylphosphate, 1 mM Na3VO4, 0.1 mM NH4MoO3) and protease inhibitors (10 U/ml aprotinin, 0.1 mM phenylmethylsulfonylfluoride, 0.1 mg/ml benzamidine, plus 10 mg/ml each of chymostatin, pepstatin, antipain, and leupeptin].

Sonicate for 30s on ice

Centrifuge at 100,000 x g for 20 min in TLA 100.3

Resuspend membrane pellets in precipitation buffer (PB = lysis buffer plus 100 mM NaCl).

Add SDS to 0.2%.

Incubate at 60C for 5 min

Add Triton X-100 to 1%.

Briefly sonicate samples on ice (no foam!)

Spin out insoluble material by ultracentrifugation (100,000 x g, 20 min).

Collect supernatant.

Add ~50 uL 1:1 slurry of BSA-blocked Ultralink-neutravidin beads (Pierce).

Rotate in cold room for 2hr.

Spin for 1-2 min at 5000rpm.

Wash with PB + 1% Triton

Repeat spin/wash four more times.

Elute biotinylated membrane proteins by boiling in SDS-PAGE.

Notes:

Can determine completeness of biotinylation by scraping and sonicating cells directly in biotinylation reagent, pulling down with neutravidin, and examining supernatant.

All washes and rinses must be done extremely gently.

If cells lift off plate, can do biotinylation, rinses, washes at 10C. This sometimes help.

If biotinylation efficiency is not a concern, can lower the amount of sulfo-NHS-SS-biotin used.

If reversibility is not needed, can substitute sulfo-NHS-biotin.

Should assess cell integrity by measuring the biotinylation of a cytoplasmic protein (e.g., tubulin).

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All procedures should be done at 4°C using precooled reagents.

Protease inhibitors	Phosphatase Inhibitors
PMSF (0.1 mM)	EGTA (2 mM)
Aprotinin (1.5 g/ml)	NaF (50 mM)
Antipain/leupeptin (10 g/ml ea.)	sodium pyrophosphate (10 mM)
Chymostatin/pepstatin (10 g/ml ea.)	-glycerophosphate (20 mM)
Benzamidine (0.1 mg/ml)	para-nitrophenylphosphate (PNPP) (1 mM)
	microcystin LR (optional) (1 M)
	Sodium orthovanadate (1 mM)
	Ammonium molybdate (0.1 mM)

1. Protocol assumes 60 mm plates.

2. Scrape cells into 250-500 ul of HEPES-buffered sucrose (0.32 M sucrose, 4 mM HEPES pH7.4).

3. Collect scraped cells and homogenize in a motor driven glass-teflon homogenizer at ~900 rpm (10-15 strokes). Never use polytron.

4. Pool samples and centrifuge the homogenate (Hom.) at 800-1000 x g at 4°C to remove the pelleted nuclear fraction (P1).

5. Spin resulting supernatant (S1) at 10,000 x g for 15 min (9200 rpm in SL50T rotor or 15,000 rpm in microfuge) to yield the crude synaptosomal pellet (P2).

6. Resuspend pellet (P2) in 1 ml of HEPES-buffered sucrose and then respin at 10,000 x g for another 15 min to yield the washed crude synaptosomal fraction (P2').

7. Lyse resulting pellet by hypoosmotic shock in 1 ml ice cold H20 (or 4 mM HEPES, pH 7.4) plus protease/phosphatase inhibitors and three strokes of a glass-teflon or eppendorf tube homogenizer

8. Rapidly adjust to 4 mM HEPES using 1 M HEPES, pH 7.4 stock solution.

9. Mix constantly in cold room for 30 min to ensure complete lysis.

10. Centrifuge lysate at 25,000 x g for 20 min (14,500 rpm in SL50T rotor; 25,000 rpm in TLA100.3 rotor) to yield a supernatant (S3, crude synaptic vesicle fraction) and a pellet (P3, lysed synaptosomal membrane fraction). For short method, proceed to postsynaptic density isolation below. For longer, more pure method, first perform the following

11. Resuspend P3 pellet in HEPES-buffered sucrose.

12. Using a Pasteur pipet, layer the resuspended membranes on top of a discontinuous gradient containing 0.8 to 1.0 to 1.2 M sucrose (top to bottom; equals 27%/34%/41%) in a clear tube. Don't forget to add protease/phosphatase inhibitors to the sucrose solutions! Always use a Pasteur pipet to pour layers and add sample.

13. Centrifuge the gradient at ~150,000 x g for 2 hr in a swinging bucket rotor (30,000 rpm in SW41 Ti; 36,000 rpm in SW50.1 Ti; 42,000 rpm in TLS-55; 28,0000 rpm in SW28).

14. Recover synaptic plasma membranes in the layer between 1.0 and 1.2 M sucrose.

15. Dilute to 0.32 M sucrose by adding 2.5 volumes of 4 mM HEPES pH 7.4.

16. Pellet by centrifugation at 150,000 x g for 30 min. 42,000 rpm in 70.1 Ti; 55,000 rpm in TLA 100.3

17. Resuspend resulting pellet (SPM) in PBS (pH 7.4) or (50 mM HEPES pH 7.4, 2 mM EDTA) with protease and phosphatase inhibitors.

18. Can store at -80°C.



Postsynaptic Densities
For references, see Carlin et al. (1980) and Cho et al. (1992).

1. Resuspend synaptic plasma membranes prepared as above in 500 uL of ice cold 50 mM HEPES pH7.4,

2 mM EDTA, plus protease/phosphatase inhibitors. Add Triton X-100 to 0.5% 2. Rotate in cold room for 15 min.

3. Centrifuge at at 32,000 x g for 20 min (22,000 rpm in 70.1 Ti; 28,000 rpm in TLA 100.3) to obtain the PSD-1T pellet.

4. Resuspend PSD-1T in 100 ul ice-cold 50 mM HEPES pH7.4, 2 mM EDTA plus protease/phosphatase inhibitors). Save aliquot.

5. To half of the remaining resuspended pellet, dilute to 500 ul with 50 mM HEPES pH7.4, 2 mM EDTA, plus protease/phosphatase inhibitors and add Triton X-100 to 0.5% and rotate in cold room again for 15 min.

6. Centrifuge at 200,000 x g for 20 min to obtain the PSD-2T pellet. 50,000 rpm in 70.1 Ti; 65,000 rpm in TLA 100.3.

7. In a separate experiment resuspend the second half of the PSD-1T pellet and incubate for 10 min in ice-cold 3% sarcosyl (same as N-lauroyl sarcosine) in 50 mM HEPES pH7.4, 2 mM EDTA, plus protease/phosphatase inhibitors.

8. Centrifuge at 200,000 x g for 1 hr to obtain the PSD-1T+S pellet.

9. All pellets can be resuspended in small volumes of PBS or 50 mM HEPES pH7.4, 2 mM EDTA plus protease/phosphatase inhibitors or SDS-PAGE sample buffer.

10. For PSD-2T pellets, may need some SDS (0.2%) to dissolve completely.

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This protocol is for preparing a large batch of PDL-coated coverslips for primary neuron or cell culture. It eliminates the need for a separate, manual coating step, does not require any UV time in the hood, saves a great deal of work, and gives you a ready supply of coverslips for use any time.

1) Rinse ~100-500 coverslips briefly 1x in 100% EtOH.

2) Shake gently 1-2 days in 100% EtOH at RT in autoclaved, covered or sealed flask or jar. Make sure coverslips are swirling.

3) In a sterile hood, rinse 4x in autoclaved water, swirling each time.

4) Submerge in 0.1 mg/ml PDL. This can be done in a 50 ml tube or in the same flask or jar. The container should be sterile and then closed. Swirl to disperse coverslips and allow them to contact the PDL solution.

5) Shake, swirl, or rock for 2 days. 200 coverslips in a 50 ml tube on the tube rotator set to very slow rotation in the cold room works well. The coverslips may bunch up eventually, but nonetheless work very well for culturing.

6) Rinse 5x with autoclaved water in the hood, swirling each time to free the PDL caught between stacked coverslips.

7) Suck off excess water, and store closed at 4ºC. They can also be laid in the wells and stored in plates wrapped in parafilm or foil.

The coverslips are at this point sterile and ready to use ?simply lay them in the wells and plate cells when needed.

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