Cell Biology Protocols

190 FRACTIONATION OF SUBCELLULAR MEMBRANES

centrifugation conditions. This can only be determined by trial and error.

8 Use of other centrifugation conditions (shorter times at higher RCFs for example) should be checked against the recommended parameters for their efficacy.

9 For gradient collection low-density end first, use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Maxidens to the

bottom of the tube or the Labconco Auto Densi-flow device to aspirate from the meniscus. For collection dense-end first, use tube puncture. More information about gradient collection can be found in ref. 26.

10 In this gradient the density of plasma membrane, lysosomes, recycling endosomes and early endosomes increases in that order[96]. Some other published papers that have reported the use of similar gradients are given in Table 5.5.

PROTOCOL 5.15

Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver [102, 103]

Reagents

Homogenization medium (HM): 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 1

OptiPrep

OptiPrep diluent (OD): 0.25 M sucrose, 6 mM EDTA, 60 mM Tris-HCl, pH 7.4

Working solution (WS) of 50% iodixanol: 5 vol. of OptiPrep + 1 vol. of OD.

Add protease inhibitors to HM and OD as required

Equipment

Gradient collector 2

High-speed centrifuge with swingingbucket for 40–50 ml tubes

Syringe (2 ml) with metal cannula 3

Ultracentrifuge with a vertical or near vertical rotor capable of approx. 350 000g (e.g. Beckman VTi65.1 or NVT65) with 11.2 ml Optiseal tubes 4

Procedure 5

Carry out ligand binding, uptake and processing, as required. Subsequently all operations must be carried out at 4 ◦C.

1.Homogenize the liver in HM (approx. 4 ml per g of tissue) as described in steps 4–6 of Protocol 4.6.

2.Centrifuge the homogenate in a swing- ing-bucket rotor at 3000gav for 10 min. The pellet may be washed with HM

if necessary and the two supernatants combined. 6

3.Make a 20% iodixanol solution (ρ = 1.127 g/ml) by diluting WS with HM.

4.Dilute the 3000g supernatant with WS 3 : 1 (v/v), so the final iodixanol concentration is 12.5% (w/v).

5.Transfer approx. 9 ml of the suspension to the Optiseal tube for the vertical or near-vertical rotor.

6.Underlay with 1.5 ml 20% iodixanol and overlay with HM to fill the tube. 7

7. Centrifuge at approx. 350 000gav for

1.5 h (slow acceleration to 800 rpm). Use slow deceleration (or no brake) from 800 rpm. 8

8.Collect the gradient, low-density end first, by upward displacement or aspiration from the meniscus or dense end

first by tube puncture, in approx. 0.5 ml fractions and analyse as required. 9

9.If it is necessary to remove cytosolic proteins from the fractions and/or to

192 FRACTIONATION OF SUBCELLULAR MEMBRANES

concentrate them, dilute with an equal volume of buffer and sediment the membranes at approx. 350 000g for 15 min. 10

Notes

This procedure will take approx. 3–4 h (excluding the operations prior to homogenization).

1 The inclusion of EDTA in this buffer removes ligand bound to the plasma membrane so that only intracellular compartments are labelled.

2 Either the Beckman Fraction Recovery System or the Labconco Auto Densiflow is suitable. For more information see ref. 26.

3 Metal ‘filling’ cannulas (i.d. 0.8–1.0 mm) can be obtained from any surgical equipment supplies company.

4 The sedimentation path length of the tube should be <24 mm. The protocol provides centrifugation times and g-forces for 11 ml Optiseal tubes for the Beckman VTi65.1; they may need to be optimized to produce the required iodixanol density gradient in other rotors. Smaller volume rotors can be used with little or no modification to the protocol, but larger volumes may require significantly longer centrifugation times. For more information on the formation of self-generated gradients see ref. 26. Although tubes other than Optiseal tubes may be used, the latter are the easiest of any sealed tube type for gradient unloading.

5 Although this fractionation protocol was used to study the endocytosis of neogalactosylalbumin by the perfused rat liver, it may also be used to analyse

the internalization of other ligands by, for example, isolated hepatocytes.

6 Although washing of the pellet will maximize the recovery of endosomes, it may make the volume of the post-heavy mitochondrial supernatant inconveniently large.

7 If a near-vertical rotor is used, the 20% iodixanol cushion may be omitted, it is present in tubes for vertical rotors to prevent any dense particle reaching the wall of the centrifuge tube; likewise the overlay may also be omitted in near-vertical rotors.

8 Slow acceleration is not needed if the dense cushion and overlay are omitted with near-vertical rotors.

9 For gradient collection low-density end first, use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Fluorinert (Sigma Aldrich) or Flutec-Blue (F2 chemicals Ltd, Preston UK) to the bottom of the tube or the Labconco Auto Densi-flow device to aspirate from the meniscus. For collection denseend first, use tube puncture. More information about gradient collection can be found in ref. 26. Under the recommended conditions, the gradient contains a central shallow region to separate light and dense endosomes. Lysosomes band in the sharp gradient formed at the bottom of the tube, while mitochondria and peroxisomes band below the lysosomes. For a more linear gradient use longer centrifugation times. To subfractionate the early clathrin-coated vesicles and the plasma membrane, which tend to have a high density, the starting concentration of iodixanol should be increased to 15 or 17.5%. To analyse more effectively

the low-density endosomes, the starting concentration of iodixanol might be reduced to 10%.

10 By removing cytosolic proteins after fractionation, endosomes need not be pelleted and resuspended prior to separation. Small volume open-topped thick-walled tubes for a micro-ultra

PROTOCOL 5.15

193

centrifuge are a convenient way of recovering sedimented membrane fractions. Do not use more than 15 min at 350 000g or 1–1.5 h at 100 000g, otherwise sedimentation of the iodixanol molecules themselves may interfere with pellet formation.

PROTOCOL 5.16

Polysucrose–Nycodenz gradients for the analysis of dense endosome–lysosome events in mammalian liver [104–108]

Reagents

Nycoprep Universal

Homogenization medium (HM): 0.25 M sucrose, 1 mM MgCl2, 10 mM TESNaOH, pH 7.4

Nycoprep Universal diluent (NUD): 6 mM EDTA, 60 mM TES-NaOH, pH 7.4

Gradient diluent (GD): 0.25 M sucrose, 1 mM EDTA, 10 mM TES-NaOH, pH 7.4

Nycodenz working solution (50%, w/v): mix 5 vol. of Nycoprep Universal with 1 vol. of NUD

45% (w/v) Nycodenz : mix 4.5 vol. of Nycodenz working solution with 0.5 vol. of 1 mM EDTA, 10 mM TESNaOH, pH 7.4 (use NUD diluted with 5 vol. of water)

20% (w/v) Nycodenz : mix 2 vol. of 45% Nycodenz with 2.5 vol. of GD

20% (w/v) polysucrose in GD 1 2

Equipment

Gradient collector 3

High-speed or refrigerated low-speed centrifuge with swinging bucket for 40–50 ml tubes

Syringe (2 ml) with metal cannula 4

Ultracentrifuge and vertical rotor with a tube volume of approx. 36 ml (e.g. Beckman VTi50 rotor) 5

Procedure 6

Carry out ligand binding, uptake and processing, as required. Carry out all operations at 0–4 ◦C.

1.Perfuse with the liver with HM until the lobes are well blanched. 7

2.Homogenize the liver in HM (approx. 4 ml per g of tissue) as described in steps 3 and 4 of Protocol 4.7.

3.Centrifuge the homogenate at 2000g for 10 min to sediment cell debris, nuclei and most of the heavy mitochondria.

4.Transfer 12.5 ml of the 20% polysucrose to 36 ml Optiseal tubes for the VTi50 rotor, then using a syringe and

metal cannula underlayer with 12.5 ml of 20% Nycodenz and 4 ml of 45% Nycodenz solution. 8 9

5.Layer the 2000g supernatant on top, to fill the tube, as specified by the manufacturer.

6. Centrifuge at 200 000g for 1 h, using a slow acceleration and deceleration programs up to and below 2000 rpm (alternatively turn off the brake below 2000 rpm).

7.Harvest the gradient in a series of equal volume fractions prior to analysis. Lysosomes band at the 45/20%

Nycodenz interface, very dense endosomes at the 20% polysucrose/20% Nycodenz ; all other endosomes band at the sample/20% polysucrose interface. 10

Notes

1 Polysucrose is available commercially under this name (Axis-Shield and its distributors) or under the trade name Ficoll (Amersham Pharmacia Biotech and its distributors). When making up solutions of these high

molecular	weight sucrose polymers,
it is better		to add	small	aliquots
(2–3 ml)	of	the	solvent	to the

weighed-out powder, using a glass rod to mix well after each addition.

2 Ellis et al. [104] made up a more concentrated Ficoll solution in water (1 ml per g) before dialysing it for 2 h against a large volume of water and adjusting it to the appropriate concentration in 0.25 M sucrose, 1 mM EDTA, 10 mM TES-NaOH, pH 7.4. It is probably easier to make up a 25% (w/v) polysucrose in 0.25 M sucrose, 1 mM EDTA, 10 mM TES-NaOH, pH 7.4 and to dialyse it against the same medium, before checking and adjusting the volume to make it 20% with respect to polysucrose.

3 Either the Beckman Fraction Recovery System or the Labconco Auto Densi-flow is suitable. For more information see ref. 26.

4 Metal ‘filling’ cannulas (i.d. 0.8–1.0 mm) can be obtained from any surgical equipment supplies company.

5 If a vertical rotor is unavailable, either a fixed-angle or a swinging-bucket rotor may be used, but the longer sedimentation path length of these

PROTOCOL 5.16

195

rotors will require longer centrifugation times.

6 This methodology applies to rat liver and although in principle it may be applied to other tissues and cultured cells, in view of the functional uniqueness of liver, it is likely that some optimization of the gradient and centrifugation conditions may be necessary.

7 See ref. 104 for more information about liver perfusion.

8 A simplified method for separating lysosomes and endosomes in which the 20% polysucrose layer was omitted has also been used.

9 More detailed analysis of the light and dense endosomes may be carried out on continuous 1–22% polysucrose gradients (with a 45% Nycodenz cushion) using approx. the same gradient volume, rotor and centrifugation conditions. The material containing lysosomes and dense endosomes at the cushion interface may be reanalysed in 0–35% or 0–45% Nycodenz gradients; see refs 104–108 for more details. Analysis of in vitro systems to study the transfer of molecules between endosomes and lysosomes has also been carried out in 0–35% Nycodenz gradients using a swinging-bucket rotor with a tube volume of approx. 14 ml, again at approx 200 000g for 1 h [109–111].

10 For gradient collection low-density end first, use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Fluorinert (Sigma Aldrich) or Flutec-Blue (F2 chemicals Ltd, Preston UK) to the bottom of the tube or the Labconco Auto Densi-flow device to aspirate from

196 FRACTIONATION OF SUBCELLULAR MEMBRANES

the meniscus. For collection denseend first, use tube puncture. More information about gradient collection can be found in ref. 26.

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