Cell Biology Protocols

Reagents

Ammonium persulfate (10% w/v, freshly made up)

Reservoir buffer: 0.192 M glycine, 25 mM Tris, 0.1% (w/v) SDS

10% (w/v) SDS

Sample buffer (SB): 4% SDS, 0.002% bromophenol blue, 20% glycerol, 10% 2-mercaptoethanol, 125 mM Tris-HCl, pH 6.5 1

Separating gel buffer: 1.5 M Tris-HCl, pH 8.8

Stacking gel buffer: 1.0 M Tris-HCl, pH 8.8

Stock acrylamide (30% T, 2.7% C): 20.2 g acrylamide, 0.8 g bisacrylamide in 100 ml distilled water Deionize with 1 g Amberlite MB-1 monobed resin per 100 ml for 2 h. Store in an amber bottle at 4 ◦C after filtering

N,N ,N ,N -tetramethylenediamine (TEMED)

20% (w/v) trichloroacetic acid (TCA) Water-saturated isobutanol

Equipment

Commercial gel cassette system, power pack, etc.

Narrow-ended tips for automatic pipette Vacuum pump and side-arm flask

1.Assemble the gel mould cassette according to the manufacturer’s instructions.

2.Make a 10% T separating gel by mixing: 33.3 ml of acrylamide stock, 25 ml of 1.5 M Tris-HCl, pH 8.8,

40.5 ml of distilled water and 150 µl of ammonium persulfate. 2

3.Degas the solution using the vacuum pump.

4. Carefully add 1.0 ml of SDS and

50 µl of TEMED; mix by gentle swirling.

5.Pour the gel solution carefully into the cassette to about 1 cm below the position of the comb.

6.Overlayer the gel with a small volume of the water saturated isobutanol.

7. Allow the gel to polymerize for

2–16 h at room temperature. 3

8.Prepare the stacking gel solution from: 3 ml of acrylamide stock, 3.75 ml of 1.5 M Tris-HCl, pH 6.8, 22.8 ml of distilled water and 135 µl of ammonium persulfate.

9.After degassing, carefully add 375 µl of SDS and 30 µl of TEMED; mix by gentle swirling.

10.Fill the cassette with the stacking gel solution and carefully insert the comb.

12.Remover the comb and set up the cassette in the electrophoresis apparatus according to the manufacturer’s instructions.

13.Add 1 vol. of sample buffer to 1 vol.

of membrane suspension and heat at 100 ◦C for 3 min.

14.Apply the samples into the wells of the stacking gel with the micropipette; 20–100 µl of the sample is commonly

used, depending on the well size and the protein concentration. 4

15.Run the gels at constant current. Standard 1.5 mm thick gels can be run at 15 mA per gel overnight.

16.Remove the gels from the cassette,

fix in TCA if necessary and stain as required, unless the gels are going to be electroblotted (see Protocol 5.11 ). 5 6 7

Notes

1 The glycerol may be omitted if dense gradient fractions containing sucrose, Nycodenz or iodixanol are to be used directly. For membrane fractions in 0.25 M sucrose include the glycerol.

2 This 10% T gel is a useful general purpose gel for resolving most proteins in the Mr range 20 000–150 000; for resolution of higher Mr proteins use a 5% T gel, for lower Mr values use a 15 or 20% T gel.

3 Polymerization overnight is often convenient.

4 The amount of sample loaded on to each track of a standard SDS-PAGE gel will vary with the sample and will depend on the staining technique used. With Coomassie blue staining the load should be 50–100 µg of protein; with silver staining, 10–50 µg of protein is normally sufficient.

5 Coomassie blue stock: 0.125 g Coomassie brilliant blue in methanol-water (405 : 520 v/v); add 7 ml of glacial acetic acid in 100 ml before use. Destain in the same solvent mixture.

6 Staining is enhanced if the gels are first rinsed with water to remove some of the SDS.

7 For glycoproteins use periodic acidSchiff or Alcian blue [28, 30].

PROTOCOL 5.11

Semi-dry blotting

Reagents

Transfer buffer: 20 mM Tris, 150 mM glycine, pH 8.3. 1

Equipment

Filter paper (Whatman 3 mM) cut to size of gel

Rocker

Semi-dry apparatus with high current capability power supply (e.g. Bio-Rad PowerPac HC)

Transfer membrane (nitrocellulose) cut to size of gel

Procedure [29, 30] 2

1.Place gel from cassette (see Protocol 5.10 ) in transfer buffer for 30 min. 3

2. Soak 12 sheets of filter paper and the nitrocellulose membrane in transfer buffer. 3

3.After allowing excess buffer to drain off each paper and gel, place six sheets of the filter paper, the membrane, the gel and six more sheets of filter paper on the anode plate of the semi-dry apparatus.

4.Place the cathode in position.

5.Transfer the proteins at 0.8 mA/cm2 for 40 min.

6.Blotted proteins are normally detected by antibody binding using a primary antibody to a membrane protein, peroxi- dase-conjugated rabbit IgG as secondary antibody followed by enhanced chemiluminescence using one of a variety of commercially available kits. See Protocol 5.12 and refs 29 and 30 for more information.

Notes

The procedure will take approx. 2 h.

1 An alternative is to use an anode buffer of 60 mM Tris-HCl, pH 9.6, 40 mM CAPS, 15% methanol and a cathode buffer of 60 mM Tris-HCl, pH 9.6, 40 mM CAPS, 0.1% SDS.

2 Carry out all reactions at room temperature and handle all materials with gloved hands.

3 If the two-buffer system is used, soak the gel and six sheets of filter paper in the anode buffer and the other six sheets in the cathode buffer.

Reagents

Diaminobenzidine (DAB): 0.05% (w/v) DAB, 0.01% (v/v) H2O2 in PBS

ECL electroblotting detection reagents; a variety of kits are commercially available and very convenient to use

Phosphate buffered saline (PBS): 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2 PO4 in 1 litre of water

PBS-T: 0.05% Tween 20 in PBS

PBS-TM: 3% (w/v) non-fat dried milk in PBS-T

Primary (monoclonal) antibody for detecting membrane antigens

Secondary antibody reagent: e.g. peroxi- dase-conjugated rabbit IgG; dilute this 1 : 1000 in PBS-TM

1.Remover the membrane carefully from the semi-dry blotter and incubate in a plastic bag with PBS-TM for 1 h to block non-specific binding sites on the membrane.

2.Transfer the membrane to a suitable dilution (e.g. 1 : 1000–1:10 000) of the

monoclonal antibody and incubate for 1 h. 4

3.Wash the membrane three times in PBS-T.

4.Incubate for 1 h with the peroxidaseconjugated secondary antibody.

5.Wash the membrane three times in PBS-T.

6.Mix the ECL reagents as recommended by the manufacturer and incubate with the membrane (0.125 ml/cm2 membrane) for 1 min.

Equipment

Cling-film

Detection film (blue-light sensitive) and suitable photoradiography cassette 1 2

Glass plate

Plastic bags 3

Rocker

Procedure [29, 30]

Carry out all operations at room temperature and use gloved hands.

7.Drain excess solution from the membrane; place on a glass plate (protein

side uppermost) and cover with clingfilm to prevent drying out. 5

8.Expose the membrane to the film in the dark. 6

9.Develop the film as recommended by the manufacturer.

10.To visualize the proteins after completion of the ECL exposure, wash the membrane twice for 15 min in PBS-T and stain with the DAB solution for 10 min.

Notes

1 Consult local manufacturer’s instructions and ref. 30 for details.

2 Rather than use the traditional film method, the blot can also be detected via a gel documentation system such as a Bio-Rad ChemiDoc XRS or a VersaDoc imager. The convenience of this method and the production of a digital image make this an increasingly popular choice.

3 Compared to plastic boxes, small volume plastic bags make more economical use of often expensive or sparingly available antibodies.

4 Use the minimum volumes permitted by the bag.

5 Avoid all air bubbles and creasing of the membrane.

6 Times between 15 s and 60 min are common. Check the manufacturer’s instructions.

(a) Mammalian cells [91, 92]

Reagents

OptiPrep

OptiPrep diluent (OD): 140 mM KCl, 60 mM Hepes-KOH, pH 7.2 containing either 15 mM Mg(OAc)2 or 12 mM EGTA and 6 mM DTT 1 2

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

WS diluent: 140 mM KCl, 10 mM HepesKOH, pH 7.2 containing either 2.5 mM Mg(OAc)2 or 2 mM EGTA and 1 mM DTT 1 2

Include protease inhibitors in solutions as required

2.Aspirate the supernatant and adjust it

to 30% (w/v) iodixanol by thorough mixing with WS. 4

3.Prepare solutions containing 25 and 5%

(w/v) iodixanol by diluting WS with WS diluent. 5

4.In tubes for the swinging-bucket rotor layer 2 ml each of the crude vesicle

fraction in 30% iodixanol and the 25% iodixanol. 6

5.Fill the tube by overlayering with 5% iodixanol. 7

6.Centrifuge at approx. 250 000g for 3 h; collect the vesicles which band at the top interface and the bottom layer containing cytosolic proteins. Alternatively

the gradient may be collected in a number of equal volume fractions. 8

Equipment

Gradient collector (optional)

Low-speed refrigerated centrifuge or microcentrifuge (in cold room)

Ultracentrifuge with swinging-bucket rotor for approx 5 ml tubes

Procedure

Carry out all operations at 0–4 ◦C.

1.Remove cells from the vesicle-contain- ing suspension either in a microcen-

trifuge or by centrifugation at 1000g for 5 min in a low-speed centrifuge. 3

(b) Bacteria [93] 9

Reagents

OptiPrep

OptiPrep diluent (OD): 3.0 M KOAc, 30 mM Mg(OAc)2,, 300 mM HepesKOH, pH 7.6 10

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

Suspension buffer (SB): 0.5 M KOAc, 5 mM Mg(OAc)2,, 50 mM Hepes-KOH, pH 7.6

WS diluent: 0.125 M sucrose in SB 10

Include protease inhibitors in solutions as required

Equipment

Ultracentrifuge with swinging-bucket rotor for approx. 5 ml tubes

Procedure

1.Suspend the crude vesicle preparation in SB.

2.Adjust the suspension to 44% iodixanol by addition of WS.

3.Transfer to tubes for the chosen swing- ing-bucket rotor.

4.Dilute WS with WS diluent to give a 30% iodixanol solution.

5.Overlayer the sample with approx. 5 vol. of 30% iodixanol. 6

6.Fill the tubes by overlayering with SB

and centrifuge at approx 170 000g for 3 h. 7

7.Either collect the gradient in three or four fractions or harvest the vesicle band at the top interface and the soluble

protein fraction (original sample zone at the bottom of the tube). 8

Notes

1 The preparation of a 50% iodixanol working solution using OptiPrep and OD maintains the concentration of buffer and Mg(OAc)2 (or EGTA and DTT) constant through the gradient when WS is diluted with WS diluent. The buffer and additives may be changed to suit the operator’s requirements; as long as their concentration in the gradient is no more than approx. 20 mM, they can be present in the OD at 6× the required concentration.

The KCl concentration in the OD is not similarly raised; if it were, gradient solutions would be highly hyperosmotic and affect the density of the vesicles.

2 The MgOAc was used by Love et al. [92] in the separation of vesicles and cytosol from permeabilized cells and the DTT/EGTA used by Scheiffele et al. [91] in the separation of budded vesicles and cytosol.

3 If the requirement is to separate the cytosol and total microsomes from a homogenate it is probably advisable to centrifuge the homogenate at 15 000g for 15 min to remove all of the larger organelles (nuclei, mitochondria, lysosomes, peroxisomes) before adjusting the density of the supernatant.

4 Rather than adjusting the crude vesicle fraction to 30% iodixanol by the addition of a 50% iodixanol WS, Love et al. [92] first pelleted the vesicles and then suspended them directly in 30% iodixanol. Either strategy should be effective.

5 Most published protocols use very similar discontinuous gradient strategies; it is worth noting, however, that Joglekar et al.[94] used a continuous 5–25% iodixanol gradient to separate COP-coated vesicles (budded from the ER/Golgi) from the cytosol also by flotation.

6 The procedure can be scaled down or up as required to suit the sample volume; change the volume of the solution above the sample proportionally; the small volume of low-density solution on top need not be increased.

7 As with all flotation methods a small volume of buffer or 5–10% iodixanol is always layered on top of the lowdensity barrier to prevent banding of the vesicles at an air/liquid interface.

8 See ref. 26 for more information on gradient fractionation. Small volume gradients may be divided into three of four zones simply by very careful aspiration using an automatic pipette.

9 The same flotation strategy has been applied to lysed yeast spheroplasts; the lysate is adjusted to 37% (w/v) iodixanol; layered beneath 30, 25, 19 and 0% iodixanol; and centrifuged at 75 000gav for 4 h to separate cytosolic and vacuolar fractions [95].

10 The preparation of a 50% iodixanol working solution (WS) using OptiPrep and OD, maintains the concentration of buffer KOAc and Mg(OAc)2 constant through the gradient when WS is diluted with WS diluent. The buffer and additives may be changed to suit the operator’s requirements; they can be present in OD at 6× the required concentration as long as their final concentration in the gradient does not significantly alter its osmolality.

Reagents

OptiPrep

Optiprep diluent (OD): 235 mM KCl, 12 mM MgCl2, 25 mM CaCl2, 30 mM EGTA, 150 mM Hepes-NaOH pH 7.0

40% Iodixanol working solution (WS): 2 vol. of OptiPrep + 1 vol. of OD

WS diluent: 78 mM KCl, 4 mM MgCl2, 8.4 mM CaCl2, 10 mM EGTA, 50 mM Hepes-NaOH pH 7.0

Homogenization medium (HM): 0.25 M sucrose, 78 mM KCl, 4 mM MgCl2, 8.4 mM CaCl2, 10 mM EGTA, 50 mM Hepes-NaOH pH 7.0 1

Protease inhibitors should be added to the HM and WS diluent as required

Equipment

Ball-bearing homogenizer, syringe with 25-gauge needle or tight-fitting Dounce homogenizer (Wheaton type A) or other suitable device 2

Gradient collector 3

Gradient maker (two-chamber) or Gradient Master

Low-speed refrigerated centrifuge with swinging-bucket rotor for 15 ml tubes

Phase contrast microscope

Ultracentrifuge with swinging-bucket rotor for 13–17 ml tubes

Procedure

Following any experimental procedures to allow binding, uptake and processing of a ligand or other functional manipulations, all operations must be carried out at 4 ◦C.

1.Remove any unbound ligand or other components from the cell surface by washing the cell monolayer twice in any

solution compatible with the study and then scrape the cells in 1 ml of HM. 4

2.Homogenize the cells using a ballbearing homogenizer (cell cracker); four passes of the cell suspension should be sufficient. Check for adequate homogenization by phase contrast microscopy. If such a device is not available then use several passages

through a fine gauge syringe needle or a Dounce homogenizer. 5

3.Centrifuge the homogenate in a swing- ing-bucket rotor at 1000g for 5 min to pellet the nuclei and any cell debris. The pellet may be washed with HM if necessary and the two supernatants combined.

4.Prepare the lowand high-density gradient solutions of 5 and 20% (w/v) iodixanol by diluting WS with the WS diluent.

5.In tubes for the swinging-bucket rotor prepare 11–14 ml 5–20% iodixanol gra-

dients using either a two-chamber gradient maker or a Gradient Master. 6

6. Layer the 1000g supernatant(s) on top of the gradient and centrifuge at 90 000gav for 18–20 h. 7 8

7.Collect the gradient in approx. 0.25 ml fractions either by upward displacement with a dense liquid or by tube puncture or by aspiration from the meniscus. The delivery of the gradient into a multi-well plate via a Gilson Fraction

Collector is a very convenient mode for analysis. 9 10

8.If it is necessary to remove the

iodixanol, fractions can be pelleted at 200 000g for 20 min after dilution with 3 vol. of HM.

Notes

1 Other homogenization media may be permissible and may need modulating if other cell types are used; see ref. 35 for more details.

2 The device of choice is the ballbearing homogenizer. Sometimes a syringe needle with a lower gauge number (wider orifice) will work satisfactorily. As with the homogenization medium, the homogenization protocol will need to be tailored to

the cell type. See ref. 35 for more information.

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

4 Surface-bound transferrin, for example, is removed by washing the cells twice in 280 mM sucrose, 25 mM citric acid, 24.5 mM sodium citrate, pH 4.6 [96].

5 Up to 10 cycles with the ball-bearing homogenizer may be needed (depending on cell type and/or HM composition), up to 15 strokes of the pestle of a Dounce homogenization or up to 20 passages through a syringe needle should be sufficient. Always monitor the progress of homogenization by phase contrast microscopy.

6 Alternatively allow a discontinuous gradient of equal volumes of 5, 10, 15 and 20% iodixanol to diffuse overnight. See ref. 26 for information on making continuous gradients.

7 Satisfactory separation of endosomal compartments from other cells or tissues may require modulation of either the density gradient or the