1. Wash 10 rat brains in HKM buffer (25mM Hepes pH 7.4, 125mM potassium acetate, 5mM magnesium acetate + 1mM DTT).

2. Make up to 40ml with HKM.

3. Add Calbiochem protease cocktail set III.

4. Homogenise brains in a potter homogeniser

5. Spin at 7000rpm for 20mins in an SS34 rotor.

6. Collect supernatant.

7. Ultracentrifuge at 45000rpm for 40 minutes in a 70Ti rotor.

8. Resuspend pellet in about 10ml of HKM.

9. Homogenise pellet.

10. Add an equal volume of HKM containing Ficoll (12.5% w/v) and sucrose (12.5% w/v). Mix by inversion to ensure homogeneity.

11. Spin in a 70Ti rotor at 25000rpm for 20 minutes.

12. Dilute the supernatant 1:5 in HKM.

13. Centrifuge at 35000rpm for 60 minutes in a 45Ti rotor.

14. Resuspend pellet in 15ml of HKM and homogenise.

15. Leave on ice for about one hour.

16. Spin at 13000rpm for 10 minutes to sediment insoluble material (70Ti).

17. Layer supernatant over a cushion of 8% (w/v) sucrose made up with D₂0 and HKM. Spin supernatant at 80,000gmax (25000rpm) in a swing-out rotor (SW40) for 2 hours. Resuspend the pellet in HKM to yield a suspension with an approximate concentration of 2mg/ml. Fractions can be snap frozen in liquid nitrogen and stored at —70C or used immediately.

NB. For Q-sepharose clathrin purification, pellets should be resuspended in 10mM Tris-HCl pH8.0 and incubated for 5 minutes. Pellet the vesicles at 50000rpm in a Beckman TLA (20min spin). The supernatant is highly enriched in clathrin. Dialyse the supernatant against 25mM Tris-HCl pH 8.0, 75mM NaCl, 1mM DTT. Run the supernatant over a small Q-sepharose column equilibrated in the same buffer. (This column has a 5ml bed volume and is currently equilibrated in 1M NaCl). Elute clathrin on 75-500mM continuous NaCl gradient (flow rate 1ml per minute). Collect 1ml fractions. Clathrin should be eluted with around 250mM NaCl.

Purify CCVs from 20 rat brains.

Uncoat in 1M Tris pH 7.4 + 1 mM DTT, with rotation at 4 deg. C fro 1 hour to overnight.

Pellet at 50,000rpm, 25 mins and load supernatant onto a gel filtration column (e.g. Pharmacia S200 16/60) equilibrated in 1M Tris pH 7.0, 1mM DTT.

Run at 1ml/min (Abs. 0.5) and collect 2ml fractions.

Clathrin elutes at around fraction 20.

Adaptors elute in a second peak at around fraction 26.

Clathrin-containing fractions are pooled and dialysed against HKM prior to use in assays.

Uncoat in 1M Tris pH7.0.

Separate clathrin from adaptors using sepharose-4B

Precipitate the clathrin-containing fractions using ammonium sulphate.

Resuspend the pellet in a minimum volume of 1M Tris pH7.0

Spin to remove aggregates.

Run supernatant down a Superose-6 column.

Dilayse clathrin containing fractions into depolymerisation buffer (20mM triethanolamine pH8.0, 1mM EDTA, 0.2mM mercaptoethanol).

Dilute the protein to an OD280 of 0.15 with depolymerisation buffer.

Dialyse into polymerisation buffer (0.1M MES pH6.5, 1mM EGTA, 0.5mM MgCl2).

Spin out clathrin cages and dialyse the supernatant against HKM.

Use the dialysed clathrin in monolayer recruitment assays.

Purified clathrin was preincubated in buffer G (25mM Hepes, 12.5mM potassium acetate and 5mM magnesium acetate, pH 7.1) and centrifuged at 90,000xg for 30 minutes to remove aggregates. The supernatant containing 1.4x10-10 moles of clathrin was incubated on ice with 2.8x10-10 moles of auxilin-1 and its' recombinant domains. The reaction mixture was divided into two aliquots and centrifuged at 90,000xg for 30 minutes. One aliquot was analysed by SDS-PAGE and the other by EM.
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