Cell Biology Protocols

Reagents

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

Dense sucrose solution (DSS): 2.0 M sucrose, 1 mM MgCl2, 10 mM TrisHCl, pH 7.4

Include protease inhibitors in these two solutions as required

Equipment

Dounce homogenizer (loose-fitting Wheaton type B, clearance 0.1–0.3 mm)

Low-speed refrigerated centrifuge with swinging-bucket rotor for 50 ml tubes

Nylon mesh (750 µm pore size)

Refractometer

Syringe (20 ml) with metal filling cannula

Ultracentrifuge with 37–39 ml swingingbucket rotor and suitable tubes

Procedure

Carry out all operations at 4 ◦C. The procedure is given for one liver from an adult male rat.

1.Blanch the liver of an anaesthetized

rat by perfusing about 20 ml of HM through the portal vein. 1

2.Rapidly remove and weigh the liver,

then chop finely with scissors in a beaker on ice. 2

3.Suspend the mince in the HM (approx. 4 ml/g of liver).

4.Homogenize the liver (in two batches)

in the Dounce homogenizer using 10 strokes of the pestle. 3

5.Filter the homogenate through the nylon mesh to remove connective tissue and unbroken cells; facilitate

filtration by gentle stirring with a glass rod. 4

6.Dilute the filtrate with HM to 5 ml/g liver and divide between two 50 ml

tubes for centrifugation at 280g for

5 min.

7.Aspirate the supernatant using a syringe and metal cannula. 5 6

8.Centrifuge the 280g supernatant at 1500g for 10 min; decant and discard the supernatant.

9.Resuspend the pellet in 25 ml of HM using the Dounce homogenizer (five strokes of the pestle) and mix well with 2 volumes of DSS. Adjust the

density if necessary to 1.18 g/ml by checking the refractive index (η = 1.4106).

10.Divide the suspension between two tubes for a Beckman SW 28 swingingbucket rotor and fill by overlayering with about 2 ml of HM and centrifuge at 113 000g for 1 h.

11.Collect the plasma membrane sheets, which band at the interface, using a syringe and metal cannula.

12.Dilute the suspension to 40 ml with HM and harvest the membranes by centrifugation at 3000g for 10 min.

13.Resuspend the pellets in the required volume (approx 1 mg protein/ml) of a suitable medium.

Notes

This procedure will take approx 3 h.

1 Cannulation of blood vessels of an experimental animal under anaesthesia

must be carried out by licensed and trained operators.

2 Failure to mince the liver adequately will result in difficult and irreproducible homogenization.

3 Avoid frothing during homogenization.

4 Do not squeeze the homogenate through the filter.

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

6 The pellet is very loosely packed; do not decant the supernatant.

Reagents

Ammonium molybdate: 2.5%, w/v in 2.5 M H2SO4

Buffer: 0.1 M Tris-HCl, pH 8.5

Fiske-Subbarow reducing agent (make up according to manufacturer’s recommendations)

Levamisole (1 mM): 240 µg/ml 1

Substrate: 20 mM adenosine monophosphate (AMP); 20 mM MgCl2, 0.2 M TrisHCl, pH 8.5

Trichloroacetic acid (TCA): 10%, w/v

Equipment

Microcentrifuge with 1.5 ml tubes

Plastic assay tubes (1.5 and 2.0 ml)

Spectrophotometer (visible wavelength)

Vortex mixer

Water bath set at 37 ◦C

Procedure 2

1.Dispense 0.7 ml of TCA in microcentrifuge tubes (two per sample) and keep on ice.

2.On ice, adjust a resuspended membrane pellet or gradient fraction (0.1 ml or less) to 0.5 ml with buffer in assay tubes and mix with 25 µl of levamisole if required.

3.Add 0.5 ml of substrate to all tubes.

4.Transfer 0.3 ml from each tube to one of the prepared TCA containing tubes (zero time reading) and mix.

5. Incubate the remaining samples at

37 ◦C for 30 min.

6.Transfer 0.3 ml of the suspension to the remaining TCA containing tubes and mix.

7.After a further 30 min on ice, remove

the precipitate in a microcentrifuge (1 min).

8.Transfer 0.5 ml of the supernatant to a 2 ml plastic assay tube.

9.For a reagent blank use 0.5 ml from a mixture of 0.7 ml of TCA and 0.2 ml buffer.

10.To each assay tube, add 0.6 ml of water, 125 µl molybdate solution and 50 µl of reducing agent, mixing well after each addition.

11.Heat at 37 ◦C for 20 min and then measure the absorbance at 820 nm

against the appropriate reagent blank. 3

Notes

1 Levamisole inhibits the hydrolysis of AMP by a non-specific alkaline phosphatase.

2 Enzyme assays based on the measurement of released phosphate can be a problem if care is not taken to eliminate, as far as possible, phosphate con-

Reagents

Buffer: 50 mM glycine-NaOH, pH 9.8

NaOH (0.25 M)

MgCl2 (1.0 M)

Substrate: p-nitrophenylthymidine 5 -phos-

phate (5 mM) (make up fresh)

Equipment

Microcentrifuge

Plastic assay tubes (1.5 ml)

Vortex mixer

Water bath set at 37 ◦C

Procedure

1.Prepare the assay solution by mixing equal volumes of buffer and substrate and to every 10 ml add 0.1 ml of 1 M MgCl2.

2.Add 0.5 ml assay solution to a resus-

pended pellet or gradient fraction (no more than 50 µl). 1

3.Incubate at 37 ◦C for 20 min and stop the reaction by adding 0.5 ml of NaOH solution.

4.Set up a blank for each sample in which the NaOH is added before the assay reagent (zero time sample) and a

reagent blank containing assay solution and NaOH. 2

2 min.

6.Measure the absorbance due to the p-nitrophenol at 410 nm against the reagent blank. The molar extinction coefficient of p-nitrophenol is 9620.

Notes

1 If the membrane is at a sufficiently high concentration, gradient samples may be used directly in the assay without removal of solutes such as sucrose, Nycodenz or iodixanol.

2 It may be permissible to omit a blank for each sample. The membrane should contribute no significant light scattering or absorbance at 410 nm in NaOH.

Reagents

Phosphate reagent A: to 0.75 g ascorbic acid in 25 ml 0.5 M HCl, add 1.25 ml 10% (w/v) ammonium heptamolybdate with vigorous agitation

Phosphate reagent B: 3% (w/v) sodium-m- arsenite, 2% (w/v) trisodium citrate (dihydrate) and 2% (v/v) acetic acid

4.Return to 37 ◦C and add 1.5 ml phosphate reagent B.

5.After 10 min, transfer 1.5 ml to a microcentrifuge tube and centrifuge for

1min.

6.Read absorbance of each supernatant at

850nm. 4

Substrate A: 3 mM MgCl2, 3 mM ATP, 130 mM NaCl, 20 mM KCl, 30 mM histidine buffer, pH 7.5

Substrate B: as medium A plus 1 mM ouabain (0.73 mg/ml) 1

Equipment

Microcentrifuge

Plastic assay tubes (4 ml)

Spectrophotometer (visible wavelength)

Vortex mixer

Water bath set at 37 ◦C

Notes

1 Ouabain inhibits specifically the Na+/ K+-ATPase.

2 Enzyme assays based on the measurement of released phosphate can be a problem if care is not taken to eliminate, as far as possible, phosphate contamination from the tubes and bottles used for the reagents. Glass tubes and glass containers must be acid-washed and then rinsed three times in distilled water before use. Plastic tubes used for the incubations need to be checked for absence of phosphate.

Procedure 2

1.For each sample set up two tubes containing 1 ml each of substrates A and B.

2.Bring the tubes to 37 ◦C; add 25 µl of

sample to each tube and incubate for 5–10 min. 3

3.Add 0.5 ml of phosphate reagent A to each tube and transfer to an ice-water bath for 6 min.

3 The activity of the enzyme is very variable; depending on the source of the membrane, use a time that provides a measurable release of phosphate, while the substrate concentration remains non-rate-limiting.

4 The difference in the phosphate concentration between the two incubations (with and without ouabain) provides that due to the Na+/K+-ATPase.

Homogenization buffer (spinach): 330 mM sorbitol, 10 mM Na2P4O7 (pyrophosphate), 5 mM MgCl2, 2 mM sodium isoascorbate, pH 6.5 1

Homogenization buffer (pea): 330 mM glucose, 5 mM MgCl2, 0.1% (w/v) bovine serum albumin (BSA), 0.1% (w/v) NaCl, 0.2% (w/v) sodium isoascorbate, 50 mM phosphate buffer, pH 6.5 1

Wash medium: 330 mM sorbitol, 2 mM EDTA, 1 mM MnCl2, 1 mM MgCl2, 50 mM Hepes-KOH, pH 7.6

40% (v/v) Percoll in wash medium

Equipment

Centrifuge (high speed) with swingingbucket rotor or fixed angle rotor (250 ml polycarbonate tubes) and swingingbucket rotor for 15 ml polycarbonate tubes 2

Cheesecloth

Cotton wool

Safety razor blade

Muslin

Rotating blades homogenizer: e.g. Polytron, Ultra-Turrax or Waring Blender

Pre-chill all equipment and solutions. Carry out all operations at 0–4 ◦C and use approx. 50 g of chosen plant material. 3

1.Harvest the material at the start of the light period and clean off any dirt adhering to the plant material in cold water and dry.

2.Devein the spinach leaves.

3.Chop the green leaves or aerial shoots

of peas very finely using razor blades and then suspend in the appropriate homogenization buffer (approx.

150 ml).

4.Homogenize in a Polytron at top speed for approx 5–10 s. 4

5.Prepare two filters (A) from two layers

of muslin and (B) from a sandwich of cotton wool between four layers of cheesecloth. 5

6.Position filter A over a 250 ml beaker on ice.

7.Pre-wet the filter with the homogenization buffer and squeeze the homogenate through the filter.

8.Pass the filtrate through filter B (do not squeeze).

9.Centrifuge the filtrate at 1500–6000g for 0.5–1.0 min. 6

10.Decant the supernatant and remove any material adhering to the tubes by wiping with a tissue.

11.Slowly pour a little wash medium down the side of the tube and gently swirl the contents to resuspend any broken chloroplasts, which form a loosely packed layer at the top of the pellet.

12.Decant the liquid and resuspend the remaining pellet in a few ml of wash medium, by very gentle agitation.

13.Dilute to the original volume of homogenate; repeat the centrifugation and resuspend the pellet in 5 ml of wash medium.

14.To remove damaged chloroplasts, layer the suspension over 6 ml of the

Percoll solution and centrifuge at 2000–3000gav for 1 min. 6

15.Resuspend the pelleted intact chloroplasts in wash medium.

Notes

The procedure will take approx. 1 h.

1 There is a great variety of homogenization media and choice of the appropriate medium will depend not only on the material but also on subsequent requirements. For more information, see ref. 58.

2 It is important for chloroplast intactness that the inner surfaces of the tubes are not scratched.

3 Some varieties of spinach, which have high levels of oxalate and phenolics, should be avoided (see ref. 58 for more information).

4 Because of the delicacy of the organelles, homogenization times should be kept to a minimum; it is preferable to accept lower yields in the interest of improved chloroplast integrity.

5 The muslin filter is critical as this retains the nuclei and broken chloroplasts.

6 The centrifugation conditions need to be optimized by the user.

Reagent

Acetone

Equipment

Glass centrifuge tubes, screw-capped (6 ml)

High-speed centrifuge with swingingbucket rotor for 6 ml tubes

Spectrophotometer (visible wavelength) with glass cuvettes (1 cm)

Procedure

1. To 10–100 µl of chloroplast suspension, add water to a final volume of

1ml.

2.Add 4 ml of acetone and mix well.

3.Centrifuge at 3000g for 2–3 min.

4.Measure absorbance of supernatant at

652nm. A652 × 27.8 = µg chlorophyll per ml of acetone extract.

Suspending medium (SM): 660 mM sorbitol, 4 mM EDTA, 2 mM MnCl2, 2 mM MgCl2, 100 mM Hepes-KOH, pH 7.6

Potassium ferricyanide (500 mM): make up fresh

DL-Glyceraldehyde (2 M)

NH4Cl (500 mM)

Equipment

Illuminator (150 W bulb) with heat filter 1

Oxygen electrode 2

Procedure

1.To the electrode chamber add in the following order: 1 ml SM, 0.9 ml distilled water, 0.1 ml of chloroplast suspension (Protocol 4.30 ), 10 µl ferricyanide and 10 µl glyceraldehyde.

2.Illuminate the mixture in the electrode chamber.

3.After 1 min, uncouple electron flow from proton gradient development by adding 10 µl NH4Cl.

4.Measure the rate of oxygen evolution (rate 1). 3

5.Repeat steps 1–4, but in step 1 add water to the chloroplast suspension first,

followed by SM and the other reagents (rate 2). 4

1 The heat filter may be a glass water bottle.

2 Consult the manufacturer’s handbook re operating instructions.

3 This is a measure of the rate of ferricyanide reduction by intact chloroplasts plus any broken organelles.

4 This is a measure of the rate of ferricyanide reduction by totally broken chloroplasts. The percentage of intact chloroplasts = [(rate 2 − rate 1)/ rate 2] × 100.

References

1. Graham, J. M. (1993) In: Methods in Molecular Biology, Vol. 19 (J. M. Graham and

J.A. Higgins, eds), pp. 29–40. Humana Press, Totowa, NJ.

2.Graham, J. M. (1997) In: Subcellular Fractionation – a Practical Approach (J. M. Graham and D. Rickwood, eds), pp. 1–29. IRL Press at Oxford University Press.

3.Chappel, J. B. and Perry, S. V (1954) Nature, 173, 1094–1095.

4.Bhattacharya, S. K. Thakar, J. H. Johnson,

P.L. and Shanklin, D. R. (1991) Anal. Biochem., 192, 344–349.

5.Bullock, G. Carter, E. E. and White, A. M. (1970) FEBS Lett., 8, 109–111.

6.Volkl, A. and Fahimi, H. D. (1985) Eur. J. Biochem., 149, 257–265.

7.Blobel, G. and Potter, V. R. (1966) Science, 154, 1662–1665.

8.Marsh, M., Schmid, S., Kern, H., Harms, E, Male, P., Mellman, I. and Helenius, A. (1987) J. Cell Biol., 104, 875–886.