189

The principles of isoelectric focusing in carrier ampholyte pH gradi-ents are described in part I. There are several reasons for the use ofagarose as separation medium.

. No toxic monomer solutions are needed;

. There are no catalysts which can interfere with the separation;

. No polymerization solutions have to be prepared and stored;

. The matrix has large pores;

. Therefore the separation times are shorter;

. The staining times are shorter;immunofixation can be carriedout in the gel.

There are however, a few problems with agarose:

. The gels are not absolutely free of electroendosmosis, sincenot all the carboxyl and sulfate groups are removed duringpurification.

. For these reasons agarose gels are not suited for use atextreme pH ranges (acid or basic).

. Gels containing urea are difficult to prepare, because urea dis-rupts the agarose network.

1Sample preparation

. Marker proteins pI 4.7 to 10.6 or

. Marker proteins pI 5.5 to 10.7 + 100 lL of double-distilled water.

. Meat extracts from pork, rabbit, veal and beef frozen in por-tions. Dilute before use: 100 lL of meat extract + 300 lL ofdouble-distilled water.

Method 5:

Agarose IEF

Electrophoresis in Practice, Fourth Edition. Reiner WestermeierCopyright � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-31181-5

See pages 51 and following.

No acrylamide, no Bis.

No TEMED, no APS.

Less work.

High molecular weight proteinsare not a problem.

Less frictional resistance.

The dried gel is stained.Seepage 18.

This leads to a cathodic drift ofthe gradient and water trans-port.

Agarose electrophoresis worksbest in the middle of the pHrange.

Use of rehydratable agarosegels (Hoffman et al. 1989).

Apply 10 lL

Apply 10 lL

Method 5: Agarose IEF

. Other samples:Set the protein concentrations around 1 to 3 mg/mL. Dilutewith double-distilled water. The salt concentration should notexceed 50 mmol/L.

It may be necessary to desalt with a NAP-10 column:apply 1 mL – use 1.5 mL of eluent.

2Preparing the agarose gel

Agarose gels are cast on GelBond film: a polyester film coated with adry agarose layer.

Agarose gels can be cast several ways: oxygen from the air does notinhibit gelation.

Here the gel is cast in a vertical prewarmed cassette, since this pro-duces an uniform gel layer. The following method is based on theexperience of Dr. Hans-J�rgen Leifheit, Munich (1987) whom wewould like to thank for his helpful advice.

Making the spacer plate hydrophobicA few mL of Repel Silane are spread over the inner face of the spacerplate with a tissue under the fume hood. When the Repel Silane isdry, the chloride ions which result from the coating are rinsed offwith water.

Assembling the casting cassette:

. Remove the GelBond film from the package.

Pour a few mL of water on the glass plate and place the supportfilm on it with the hydrophobic side down. Press the film against theglass with a roller (Fig. 1), the film should overlap the length of theglass plate by about 1 mm.

The spacer plate, with the gasket on the bottom, is then placed onthe glass plate and the cassette is clamped together (Fig. 2).

Before pouring the hot agarose solution, prewarm the cassette anda 10 mL pipette to 75 �C in a heating cabinet.

190

Apply 10 lL

Not to be confused withGelBond PAG film.

Unlike polymerizationof acrylamide.

Leifheit H-J, Gathof AG,Cleve H. �rztl Lab. 33 (1987)10–12.

The “spacer” is the glass platewith the 0.5 mm thickU-shaped silicone rubbergasket.This treatment only needs to becarried out once.

Identify the hydrophilic sidewith a few drops of water.

This facilitates filling thecassette later on.

This ensures that the solutiondoes not solidify immediately.

2 Preparing the agarose gel

Preparation of the agarose solution (0.8% agarose)

It is important to store agarose in a dry place because it is very hygro-scopic.

. In a 100 mL beaker:2 g of sorbitol19 mL of distilled water0.16 g of agarose IEF

. Mix and boil the solution – covered with a watch glass – untilthe agarose has completely dissolved either in a microwaveoven at the lowest setting or while stirring slower on a mag-netic heating stirrer.

. Degas to remove CO2.

. Place the beaker in a heating cabinet for a few minutes to coolthe solution down to 75 �C.

. Add 1.3 mL of Ampholine pH 3.5 to 9.5 or Pharmalyte pH 3 to10 and stir with a glass rod.Most of the commercially available solutions contain 40 %(w/v) carrier ampholytes. The product “Pharmalytes�” are

191

Fig. 1: Applying the support film with a roller.

Fig. 2: Assembling the gel cassette.

If the agarose is humid, toolittle will be weighed.

Sorbitol improves the mechan-ical properties of the gel andsince it is hygroscopic, it worksagainst the electro-osmoticwater flow.

Rapid stirring damages themechanical properties ofagarose.

It should not be too hot for thecarrier ampholytes.

Method 5: Agarose IEF

produced with a different chemistry, the concentration cantherefore not be specified. However, they are used with thesame volumes like a 40 % solution.

. Remove the cassette from the heating cabinet; draw the hotagarose solution in the prewarmed pipette and quickly releaseit in the cassette (Fig. 3).

. Let the cassette stand for 1 or 2 h at room temperature.

. Remove the clamps and take out the gel.

. Place the gel on a piece of moist tissue and store it overnightin a humidity chamber (Fig. 4) in the refrigerator, it can bekept for up to a week.

192

Fig. 3: Pouring the hot agarose solution in theprewarmed cassette.

gel

wet filter paper

Fig. 4: Agarose gel in the humidity chamber.

Avoid air bubbles. Should somebe trapped nevertheless,dislodge them with a long stripof polyester film.

The gel slowly sets.

Only then does the finalagarose gel structure form(see page 15).

3 Isoelectric focusing

Preparation of electrode solutionsIn Tab 1 a list of electrode solutions is given for respective pH intervals.

Tab. 1: Electrode solutions for IEF in agarose gels

pH gradient anode cathode

3.5 – 9.5 0.25 mol/L acetic acid 0.25 mol/L NaOH2.5 – 4.5 0.25 mol/L acetic acid 0.40 mol/L HEPES4.0 – 6.5 0.25 mol/L acetic acid 0.25 mol /L NaOH5.0 – 8.0 0.04 mol/L glutamic acid 0.25 mol/L NaOH

3Isoelectric focusing

. The gel is placed with the film on the bottom on the coolingplate at 10 �C, using about 1 mL kerosene (Fig. 6).

. Dry the surface with filter paper (Fig. 5) because agarose gelshave a liquid film on the surface.

Electrode solutions:

To reduce cathodic drift apply electrode strips made from filter paperbetween the electrodes and both edges of the gel and let them soak inthe electrode solutions. For a pH gradient from 3.5 to 9.5 these are:

Anode: Cathode:0.25 mol/L acetic acid 0.25 mol/L NaOH

. Cut the electrode strips shorter than the gel (< 25 cm);

. Soak the strips thoroughly in the corresponding solutions;

. Blot them with dry filter paper for about 1 min to removeexcess liquid;

193

Fig. 5: Drying the surface of the gel with filterpaper.

IEF must be performed at adefined constant temperaturebecause the pH gradient andthe pIs are temperaturedependent.

These are also used for pH 4.0to 6.5.

They should not protrude thesides.

Method 5: Agarose IEF

. Place the electrode strips along the edge of the gel;

. Shift the electrodes along the electrode holder so that they lieover the electrode strips (see Fig. 6);

. Plug in the cable, make sure that the long anodic connectingcable is hooked to the front.

Separation conditions

The values for current and power are valid for a whole gel. For IEF ofhalf a gel use half the values for mA and W.

. Prefocusing: 30 min at 1400 V, 30 mA, 8 W,

. The optimum point for sample application in agarose IEFdepends on the characteristic of the sample (see also method6, page 204). But most samples can be applied where men-tioned here.Place the sample applicator strip (mask) on the gel surface 2cm away from the anode.

. Sample application: apply 10 lL in each hole of the sampleapplicator .

. Desalting: 30 min at max. 150 V; the other settings remain.

. Separation: 60 min at 1500 V, 30 mA, 8 W.

194

cathodeNaOH acetic acid

sample applicatormask

anode

Fig. 6: Agarose IEF with electrode strips andsample application strip.

Make sure that the acid striplies under the anode and thebasic strip under the cathode.

The pH gradient is formedduring this step.

It may be necessary to do astep test.

Do not use any filter paper orcellulose plates for agarose IEF!

This also helps high molecularweight proteins, e.g. IgM toenter the gel

This is valid for gradients frompH 3 to 10.

4 Protein detection

During the run it may be necessary to interrupt the separation andblot the electrode strips with filter paper.

For narrower gradients, e.g. pH 5 to 8, IEF should last about 2 h,since the proteins with a low net charge must cover long distances.

. Switch off the power supply, open the safety lid, remove theelectrodes and blot the excess water from the strips;

. Continue the separation.

The proteins are then stained, immunofixed or blotted. Shouldproblems occur, consult the trouble-shooting guide in the appendix.

4Protein detection

Coomassie Blue staining

. Fixing: 30 min in 20% (w/v) TCA;

. Washing: 2 �15 min in 200 mL of fresh solution each time:10% glacial acetic acid, 25% methanol;

. Drying: place 3 layers of filter paper on the gel and a 1 to 2 kgweight on top (Fig. 7). Remove everything after 10 min andfinish drying in the heating chamber;

. Staining: 10 min in 0.5% (w/v) Coomassie R-350 in 10% gla-cial acetic acid, 25% methanol: dissolve 3 PhastGel Blue Rtablets (0.4 g dye each) in 250 mL;

. Destaining: in 10% glacial acetic acid, 25% methanol till thebackground is clear;

. Drying: in the heating cabinet.

195

glass plate

gel

filter paper

Fig. 7: Pressing the agarose gel.

Prepare all solutions withdistilled water.

First moisten the piece of filterpaper lying directly on theagarose.

Method 5: Agarose IEF

ImmunofixationOnly the protein bands, which have formed an insoluble immunopre-cipitate with the antibody are stained with this method.

Antibody solution: dilute 1:2 (or 1:3 depending on the antibodytiter) with double-distilled water and spread it over the surface of thegel with a glass rod or a pipette: use about 400 to 600 lL;

. Incubation: 90 min in the humidity chamber in a heatingchamber or incubator at 37 �C;

. Pressing: 20 min under 3 layers of filter paper, a glass plate anda weight (Fig. 7);

. Washing: in a physiological salt solution (0.9% NaCl w/v) over-night;

. Drying: see Coomassie staining;

. Staining and destaining as for Coomassie staining.

It is important to know the antibody titer, since hollow bands canappear in the middle when the antibody solutions are too concen-trated: one antigen binds to one antibody and no precipitate isformed.

Silver stainingIf the sensitivity of Coomassie staining is not sufficient, silver stain-ing is carried out on the dry gel (Willoughby and Lambert, 1983):

Solution A: 25 g of Na2CO3, 500 mL of distilled water;

Solution B: 1.0 g of NH4NO3, 1.0 g of AgNO3, 5.0 g of tungstosilicicacid, 7.0 mL of formaldehyde solution (37%), make up to 500 mLwith distilled water.

Staining: mix 35 mL of solution A with 65 mL of solution B justbefore use. Soak the gel immediately in the resulting whitish solutionand incubate while agitating until the desired intensity is reached.Rinse briefly with distilled water;

Stop with 0.05 mol/L glycerol. Remove remains of metallic silverfrom the gel and the back of the support film with a cotton swab.

Drying: air-dry.

196

All the other proteins andexcess antibodies are washedout of the gel with a NaClsolution.

First moisten the piece of filterpaper lying directly on the gel.