神经元的培养及转染(3)

Biotinylation Assay of Receptor Endocytosis with Cleavable Biotin Reagent

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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PSD Prep from High-Density Cultured Neuron

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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Large-batch Procedure for Preparing PDL-coated Coverslips

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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神经元的培养及转染(3)