Cell Biology Protocols

Reagents

Reaction buffer: 0.5% (w/v) Triton X-100, 0.05 M sodium-citrate-phosphate buffer, pH 4.3

Stop buffer: 0.25 M glycine-NaOH, pH 10 1

Substrate: 6 mM p-nitrophenyl β-D-gal-

actopyranoside in reaction buffer

Equipment

Microcentrifuge with 1.5–2.0 ml tubes

Spectrophotometer (visible wavelength) with 1 ml cuvettes

Procedure [73]

1.For each test, mix 0.5 ml of substrate with a membrane fraction pelleted in a microcentrifuge tube (see step 1 of

Protocol 4.11 ) or with up to 50 µl of a membrane suspension. 2

2.Set up controls as in step 1 and add 1 ml of stop buffer immediately. 3

3.Incubate tests at 37 ◦C for 30 min.

4.Add 1 ml of stop buffer to tests.

5.Remove any sedimentable material in a microcentrifuge (1–2 min).

6.Read the absorbance of tests vs controls at 410 nm.

Notes

The procedure will take approx. 1 h.

1 Do not allow the pH of the stop buffer to rise above 10 as this will cause hydrolysis of the substrate.

2 If the sample is sufficiently concentrated, up to 50 µl of sample can be added directly to the substrate solution. Dilute samples should first be concentrated by pelleting in a microcentrifuge.

3 If none of the samples contributes to the overall absorbance, it is permissible to use a single control without membrane suspension. There is negligible hydrolysis of the substrate at pH 4.3 in the absence of enzyme.

Reagents

Reaction buffer: 100 mM sodium acetateacetic acid, pH 4.3, 100 mM KCl, 2% Triton X-100, 5 mM DTT

Substrate: 1.7 mM 4-methylumbelliferyl- β-D-galactoside (4-MUG) in reaction buffer

3.Mix with an equal volume of substrate and incubate at 37 ◦C for 1 h. Set up

sample blanks using 0.25 ml of reaction buffer instead of substrate. 1

4.Set up also a substrate blank.

5.After 1 h measure the fluorescence. 2

Equipment

Fluorimeter, 355 nm emission, 460 nm excitation with cuvettes to take 200 µl

Microcentrifuge

Water bath at 37 ◦C

Procedure [74]

1.After dilution with two volumes of

buffer, pellet the gradient fraction in the microcentrifuge for 10 min at 0–4 ◦C.

2.Suspend the pellet in 0.25 ml of reaction buffer. 1

Notes

This procedure will take approx. 2 h.

1 It will be necessary to run a few trial assays to optimize the amount of membrane required; determine that the rate of reaction is linear with time and membrane concentration. The assay is approx. 5–10 times more sensitive than the spectrophotometric assay (see

Protocol 4.13 ).

2 The substrate blank should read no more than 10 fluorescence units against a value of at least 500 units for the test.

Reagents

OptiPrep (60% iodixanol)

Homogenization medium (HM): 0.25 M sucrose, 1 mM EDTA, 0.1% (v/v) ethanol, 5 mM Mops pH 7.2. 1

Diluent: 6 mM EDTA, 0.6% ethanol, 30 mM Mops, pH 7.2

1 M sucrose

Add protease inhibitors to HM and diluent as required

Light mitochondrial fraction prepared using HM (see Protocol 4.8)

Equipment

Dounce homogenizer (10 ml, loose-fitting, Wheaton type B)

Two-chamber gradient maker or Gradient Master

Syringe (5 ml) with metal cannula (internal diameter approx. 0.8 mm) 2

Ultracentrifuge and 30–40 ml fixed-angle rotor capable of approx. 100 000g, with thick-walled tubes of approx 30 ml capacity

Gradient collector (e.g. Beckman Fraction Recovery System)

Procedure [75]

Carry out all operations at 0–4 ◦C.

1.Make up the following gradient solutions from OptiPrep , diluent, 1 M

20% iodixanol (2 + 0.6 + 1.1 + 2.3).

3 4 5

2.Resuspend the light mitochondrial pellet in HM using a loose-fitting Dounce homogenizer (two to three strokes of the pestle). Adjust to a volume of about 0.5 ml per g of tissue.

3.Use a two-chamber gradient maker or a Gradient Master to prepare a linear gradient from 9 ml each of 20 and 40% iodixanol solutions in thick-walled polycarbonate tubes for the fixed-angle rotor and, using the syringe and metal

cannula, underlayer each gradient with 2 ml of 50% iodixanol. 6 7

4.Layer 3 ml of the suspension over each

gradient and centrifuge at 105 000gav for 1 h.

5.Allow the rotor to decelerate from 1000 rpm without the brake and collect the gradient in 1 ml fractions, dense end first. Alternatively, collect the peroxisomes that band approx. two-thirds of

the way down the gradient, using the syringe and metal cannula. 8

Notes

1 Use this HM to prepare the light mitochondrial fraction.

2 Metal ‘filling’ cannulas can be obtained from any surgical equipment supplies company.

3 The use of the four components to prepare the gradient solutions assures that each is isoosmotic.

4 Van Veldhoven et al. [75] developed this method for rat liver. Iodixanol gradients (15–45%) have also been used in vertical rotors for mouse [76] and human liver [77]. The shorter sedimentation path length of a vertical rotor permits the use of considerably lower g-forces (31 000g for 75 min). This may improve the retention of functional integrity of the organelles. On the other hand, a 4–30% iodixanol gradient at 100 000g for 17 h has been used for CV1 cells [78].

5 Gradients covering a similar density range are used with Nycodenz :

for yeast, 15–42.5% at 174 000g for 75 min [79] and 15–36% at 100 000g for 1 h [80]; and for rat liver, gradients vary from 25–50% [81] to 15–48% [82] and centrifugation conditions from 60 000g for 75 min [81] to 35 000g for 75 min [83]. Many of these methods employ a vertical rotor.

6 If neither of these devices is available, mix equal volumes of the 20 and 40% iodixanol solutions to produce an additional 30% iodixanol solution. Create a discontinuous gradient from 6 ml each of the three solutions and allow a continuous gradient to

be formed by diffusion overnight at 0–4 ◦C.

7 See ref. 18 for more information about making gradients.

8 Use either tube puncture or aspiration from the bottom of the tube to collect the gradient. See ref. 18 for more information about harvesting and analysing gradients.

Reagents

Peroxide stock: 30 mM H2O2 in T-BSA 1

Sample buffer: 2 vols. T-BSA + 1 vol. 2% Triton X-100

Titanium oxysulfate (2.25 g/l) in 1 M H2SO4 2

T-BSA: 20 mM Tris-HCl, pH 7.0 containing 0.1% (w/v) bovine serum albumin

5.After exactly 1 min, add 1.0 ml of tita-

nium oxysulfate and microcentrifuge ( 13 500g) for 2 min.

6.Allow all tubes to reach room temperature and measure the absorbance at

405 nm against a blank containing 0.54 ml of T-BSA and 1 ml of titanium oxysulfate. 5

Equipment

Microcentrifuge with 2.0 ml microcentrifuge tubes

Spectrophotometer

Procedure [84]

Carry out all procedures on ice in 2 ml microcentrifuge tubes and keep all solutions on ice.

1.Make up the assay mixture freshly by diluting 8.5 ml of stock peroxide to 100 ml with T-BSA.

2.Add 1.0 ml of titanium oxysulfate to 0.5 ml of assay mixture and measure

the absorbance at 405 nm; it should be1.5. If it is not, then adjust the H2O2 concentration accordingly.

3.Mix 10 µl of sample with 30 µl of

sample buffer; for the reagent control, use 40 µl of sample buffer. 3 4

4.Add 0.5 ml of assay mixture to all tubes; do this in batches of six tubes only.

Notes

This procedure will take approx. 1 h.

1 Keep the stock peroxide at 4 ◦C.

2 Titanium oxysulfate is highly corrosive.

3 The enzyme is very active in mammalian liver fractions: peroxisomerich fractions may have to be diluted up to 100× with sample buffer.

4 It is usually unnecessary to use a blank for each fraction; if it is deemed necessary, then set up duplicate samples (step 4) and add the titanium oxysulfate before the assay mixture to one of each pair.

5 The titanium oxysulfate reacts with the H2O2 remaining after the incubation; thus if all the substrate had been used up, the absorbance would be close to zero. The reagent control absorbance measures the substrate concentration at zero time.

Reagents

OptiPrep (60% w/v iodixanol)

Homogenization medium (HM): 0.25 M sucrose, 1 mM EDTA, 20 mM HepesNaOH, pH 7.4

OptiPrep Diluent (OD): 0.25 M sucrose, 6 mM EDTA, 120 mM Hepes-NaOH, pH 7.4

Iodixanol (50%, w/v) working solution (IWS): 5 vols. OptiPrep + 1 vol. OD 1

Add protease inhibitors to HM and OD as required

Light mitochondrial fraction prepared using HM (see Protocol 4.8)

Equipment

Dounce homogenizer (loose-fitting, Wheaton type B)

Two-chamber gradient maker or Gradient Master

Syringe (5 ml) with metal cannula (internal diameter approx. 0.8 mm) 2

Ultracentrifuge with swinging-bucket rotor (tube capacity of approx. 17 ml)

Gradient collection device for harvesting gradient low-density or high-density end first 3

Procedure

Carry out all operations at 0–4 ◦C.

1.Dilute IWS with HM to produce gradient solutions with iodixanol concentra-

tions of 19 and 27% (w/v). Keep these solutions on ice until required. 4

2.Resuspend the light mitochondrial pellet in approx 3.0 ml of HM using the loose-fitting Dounce homogenizer (two to three strokes of the pestle).

3.Adjust the suspension to 30% iodixanol by mixing with IWS.

4. Use a two-chamber gradient maker or a Gradient Master to prepare a linear gradient from 6 ml each of the two gradient solutions in tubes for the swinging-bucket rotor. 5

5.Using the syringe and metal cannula layer 3–4 ml of the suspension (step 3) beneath the gradient.

6.Layer 1–2 ml of HM on top of the gradient to fill the tube to about 3 mm from its top and centrifuge in a suitable swinging-bucket rotor at approx 70 000gav for 1.5–2 h.

7.Collect the gradient in 1 ml fractions low-density end first by upward displacement with a dense medium or by aspiration from the meniscus. Alternatively, collect dense end first by tube puncture or by carefully introducing a narrow metal cannula (connected to a

peristaltic pump) to the bottom of the tube. 3 6 7

the gradient by tube puncture, is satisfactory. The Beckman Fraction Recovery System incorporates a good tube puncture device. This can also be used for collection dense end first.

4 There are variations to the density range of the gradient used in this protocol. The 19–27% iodixanol gradient is optimal for rat liver; some other examples are summarized in Table 4.2. In nearly all cases, the sample was layered on top of the gradient rather than beneath it, even though in the latter format the resolution is probably greater.

5 If neither of these devices is available create a discontinuous gradient from 3 ml each of 19, 22, 25 and 27% iodixanol and allow a continuous gradient to be formed by diffusion overnight at 0–4 ◦C. For more information on the formation of gradients, see ref. 18.

6 For more information about the harvesting and analysis of gradients, see ref. 18.

7 A typical result with rat liver is shown in Figure 4.1.

Reagents

Homogenization medium (HM): 0.25 M sucrose, 10 mM Hepes-NaOH, pH 7.4

Gradient solutions: 13.6, 17.5, 50 and 51.5% (w/v) sucrose in 10 mM HepesNaOH, pH 7.4

Rough ER suspension medium (RSM) 70% (w/v) sucrose, 3 mM imidazoleHCl, pH 8.2

Stripping solution: 68% (w/v) sucrose, 40 mM pyrophosphate, 3 mM imidazole -HCl, pH 8.2

Light mitochondrial supernatant: Protocol 4.8 (using the above HM) as far as step 8

Include protease inhibitors in the solutions as required

Equipment

Dounce homogenizer (20–30 ml loosefitting, Wheaton type B)

Two-chamber gradient maker or Gradient Master

Syringe (5 or 10 ml) and metal cannula 1

Ultracentrifuge with 8 × 39 ml fixed-angle rotor (with open-topped thick-walled tubes) and 14–17 ml swinging-bucket rotor

1.Centrifuge the 15 000g supernatant at 100 000g for 40 min.

2.Discard the supernatant and resuspend

the microsomal pellet in 20 ml of the homogenization medium using the Dounce homogenizer. 3

3. Using a two-chamber gradient maker or Gradient Master , prepare 17.5– 50% (w/v) sucrose gradients in tubes for the swinging-bucket rotor (for 17 ml tubes make a 5 ml gradient). 4

4.Underlayer the gradient with 2 ml of 51.5% sucrose using the syringe and metal cannula.

5.Layer 10 ml of the microsomal sus-

pension on top and centrifuge at 100 000g for 16 h.

6.Harvest the smooth ER band by removing the top 15 ml of the gradient using a syringe and metal cannula and concentrate (after dilution with an equal volume of HM) by centrifugation at 100 000g for 1 h.

7.Resuspend the pellet of rough microsomes (from the gradient tube) in the residual medium (2 ml) and add 12 ml of the RSM plus 5 ml of stripping solution.

Procedure

The method is designed for approx 10 g of

rat liver. Carry out all operations at 0–4 ◦C.

2

8.Prepare 13.6–50% (w/v) linear sucrose gradients (12 ml in 17 ml tubes)

and underlay each with the 5 ml of sample. 5

9.Centrifuge at 200 000g for 2 h.

10.The stripped RER vesicles band in the bottom half of the gradient (27–50% sucrose) while peroxisomal and mitochondrial contaminants are located below this band.

11.Collect the band using a syringe and metal cannula.

12.Dilute with 2 vols. of HM and pellet at 100 000g for 1 h.

Notes

This procedure will take approx. 1.5 days.

1 Metal ‘filling’ cannulas can be obtained from any surgical equipment supplies company.

2 Although developed for rat liver the method may be used for any tissue.

3 The rough ER in the 100 000g pellet tends to make the latter gelatinous; make sure this is well dispersed.

4 If neither of these devices is available then allow a discontinuous gradient (1 ml each of 17.5, 25.5, 32.5, 40.5 and 50% sucrose) to diffuse for approx 6 h. See ref. 18 for more information about making gradients.

5 If neither a two-chamber mixer nor a Gradient Master is available, allow a discontinuous gradient (2.5 ml each of 13.6, 22.6, 31.6, 41.6 and 50% sucrose) to diffuse overnight. See ref. 18 for more information about making gradients.