3mM imidazole (1ml of 0.3M stock)

250mM sucrose (8.56g)

protease inhibitor cocktail

pH to 7.2 with KOH

Prepare 5 confluent 10cm dishes of BHK cells.

Can internalise HRP (1mg/ml, PBS++) for 10 min before scraping cells.

Prepare PNS.

Remember to include proteinase inhibitors in homogenisation buffer.

Prepare 62% sucrose (w/w) in 3mM imidazole, pH 7.4

Always check % sucrose with refractometer

Dilute PNS approx 1:1 with 62% sucrose solution to give a final sucrose value of 40.6% +- 0.5%.

Check all sucrose concentrations using the refractometer

Add 1-1.5ml of load fractions to 4.4ml kontron centrifuge tube and then carefully pipette on to top surface of tube 1.0ml of 16% sucrose followed by 1ml of 10% sucrose. Then add homogenisation buffer (HB) to fill the tube. The centrifuge tubes need to be used with adaptors, which are normally kept in Bobs lab..

Spin at 35000rpm for 1hour at 4°C.

Collect the 16%-10% early endosome enriched interface, using a capillary tube attached to a peristaltic pump. If HRP has been internalised->HRP assay on all fractions to measure the degree of enrichment.

Measure volume of endosome fraction and perform protein assay using BioRad detergent method. 30-40µg of endosomes were obtained from 5 dishes from 2 separate experiments.

Refs:

Aniento, F., Emans, N., Griffiths, G., and Gruenberg, J. (1993). Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J.Cell Biol. 268, 10463-10470.

Gorvel, J. P., Chavrier, P., Zerial, M., and Gruenberg, J. (1991). Rab 5 controls early endosome fusion in vitro. Cell 64, 915-925.