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4.1Principle

Blotting is the transfer of large molecules on to the surface of animmobilizing membrane. This method broadens the possibilities ofdetection for electrophoretically separated fractions because the mole-cules adsorbed on the membrane surface are freely available for mac-romolecular ligands, for example antigens, antibodies, lectins ornucleic acids. Before the specific detection the free binding sitesmust be blocked with substrates, which do not take part in the ensu-ing reaction (Fig. 36).

4

Blotting

blockingreagent

antibodieslectins

proteins, DNA, RNA

gel

transfer

blocking

specificligands

"probes"

blotting membrane

Fig. 36: The most important steps during blotting from electrophoresis gels.

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

In addition blotting is an inter-mediate step in protein sequen-cing and an elution method forsubsequent analyses.

4 Blotting

4.2Transfer methods

4.2.1Diffusion blotting

The blotting membrane is applied on to the gel surface as when mak-ing a replica. The molecules are transferred by diffusion. Since themolecules diffuse regularly in every direction, the gel can be placedbetween two blotting membranes thus yielding two mirror-imagetransfers (Fig. 37). The diffusion can be accelerated by increasing thetemperature, the technique in then known as thermoblotting. It ismostly used after electrophoresis in gels with large pores.

4.2.2Capillary blotting

This technique is a standard one for subsequent hybridization accord-ing to Southern (1975) (Southern blot) during DNA separations. Thetransfer of RNA on to a covalently binding film or nylon membranewhich is now known under the name Northern blot also uses thistechnique (Alwine et al. 1977).

This kind of transfer can also be used for proteins, which were sep-arated in a gel with large pores (Olsson et al. 1987). Buffer is drawnfrom a reservoir through the gel and the blotting membrane to astack of dry paper tissues by capillary force. The molecules are carriedto the blotting membrane on which they are adsorbed. The transferoccurs overnight (Fig. 38).

4.2.3Pressure blotting

Pressure blots from agarose gels cast on GelBond film are obtainedvery easily: a wet blotting membrane is laid on the gel, covered by oneor several dry filter paper sheets, a glass plate and a 1 kg weight for

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blotting membrane

diffusion

diffusion

gel

blotting membrane

Fig. 37: Bi-directional transfer of proteins bydiffusion blotting from a gel with large pores.

Quantitative transfers cannotbe achieved with this method,especially not with larger mole-cules.

Southern EM. J Mol Biol. 98(1975) 503–517.Alwine JC, Kemp DJ, Stark JR.Proc Natl Acad Sci USA, 74(1977) 5350–5354.

Olsson BG, Westr�m BR,Karlsson BW. Electrophoresis 8(1987) 377–464.

Desvaux FX, David B, Peltre G.Electrophoresis 11 (1990)37–41.

4.2 Transfer methods

100 cm2. The transfer is very fast, only a few seconds! Even multiplesuccessive and identical blots can be obtained from one single gel(Desvaux et al. 1990).

The most effective transfer from isoelectric focusing gels is pres-sure blotting according to Towbin et al.

4.2.4Vacuum blotting

This technique is mostly used instead of capillary blotting (Ols-zewska and Jones, 1988). It is important to have a controlled low vac-uum with, depending on the case, a 20 to 40 cm high water columnto prevent the gel matrix from collapsing. An adjustable pump isused since a water pump yields a vacuum that is too high and irregu-

69

1kg

buffer

filter paperblotting membrane

dry tissuepaper

agarosegel

Fig. 38: Capillary blotting, the transfer occursovernight.

buffer reservoir

porous divider

agarosegel

mask

blotting membrane

vacuum chambertovacuumpump

Fig. 39: Transfer of nucleic acids with vacuumblotting in 30 to 40 min.

Towbin H, �zbey �, Zingel O.Electrophoresis 22 (2001)1887–1893.

Olszewska E, Jones K. TrendsGen. 4 (1988) 92–94.

Also pressure can be employed;this is also called pressure blot-ting.

4 Blotting

lar. The surface of the gel is accessible to reagents during the entireprocedure. A diagram of a vacuum blotting chamber is representedin Fig. 39.

Vacuum blotting possesses some advantages over capillary blot-ting, it:

. is faster: 30 to 40 min, instead of overnight;

. is quantitative, there are no back transfers; leads to sharperzones and better resolution;

. allows faster depurination, denaturation and neutralization;

. reduces the mechanical stress on the gel;

. saves expenses for solutions and paper.

4.2.5Electrophoretic blotting

Electrophoretic transfers are mainly used for proteins SDS electro-phoresis (Towbin et al. 1979; Burnette, 1981). Only in some cases alsonucleic acids are transferred with the help of an electric field. Eitherdifferent samples are applied on a gel and analysed together on themembrane, or the antigen solution is separated across the entire gelwidth and the membrane is cut into narrow strips for probing in dif-ferent antibody solutions (e.g. patients sera).

Electrophoretic protein blotting followed by immunodetection isfrequently called “Western Blotting” following the methodical evolu-tion from “Southern” and “Northern Blotting”. The modification ofblotting proteins and probing the membrane with a non-antibodyprotein to detect specific protein-protein interactions is called “Far-Western blotting” (Burgess et al. 2000). The probing protein is thentagged with a labeled antibody for detection.

Tank blotting

Originally, vertical buffer tanks with coiled platinum wire electrodesfixed on two sides were used. For this technique, the gel and blottingmembrane are clamped in grids between filter papers and spongepads and suspended in the tank filled with buffer. The transfersusually occur overnight.

Semi-dry blotting

Semi dry blotting between two horizontal graphite plates has gainedmore and more acceptance in the last few years. Only a limited vol-ume of buffer, in which a couple of sheets of filter paper are soaked,is necessary. This technique is simpler, cheaper and faster and a dis-continuous buffer system can be used (Kyhse-Andersen, 1984; Toveyand Baldo, 1987).

70

in the tank

Gel remains in the tank.

Towbin H, Staehelin T,Gordon J. Proc Natl Acad SciUSA. 76 (1979) 4350–4354.Burnette WN. Anal Biochem.112 (1981) 195–203.

Burgess R, Arthur TM, Pietz BC.Meth Enzymol. 328 (2000)141–157.

The buffer should be cooled sothat the blotting sandwich doesnot warm up too much.

Kyhse-Andersen J. J BiochemBiophys Methods. 10 (1984)203–209.Tovey ER, Baldo BA. Electro-phoresis 8 (1987) 384–387.

4.2 Transfer methods

The isotachophoresis effect occurs here: the anions migrate at thesame speed, so that a regular transfer takes place. A system of graph-ite plates does not need to be cooled. A current not higher than 0.8 to1 mA per cm2 of blotting surface is recommended. The gel can over-heat if higher currents are used and proteins can precipitate.

The transfer time is approximately one hour and depends on thethickness and the concentration of the gel. When longer transfertimes are required as for thick (1 mm) or highly concentrated gels, aweight is placed on the upper plate so that the electrolyte gas isexpelled out of the sides. Fig. 40 shows a diagram of a semi dry-blot-ting setup.

It is also possible to perform electrophoretic transfers on two mem-branes simultaneously: Double Replica Blotting (Johansson, 1987). Analternating electric field is applied on a blotting sandwich with amembrane on each side of the gel with increasing pulse time, so thattwo symmetrical blots result.

Blotting of gels supported by films

Ready-made or self-made gels backed by support films are used moreand more often for electrophoresis of proteins and electrofocusing.These films which are inpermeable to current and buffer must beseparated from the gels so that electrophoretic transfers or capillaryblotting can be carried out. To separate the gel and the film withoutdamage an apparatus exists with a taut thin steel wire which is pulledbetween them (see Fig. 41).

71

cathode

anode

Trans-Unit

cathode buffer

gelblot membrane

anode buffer II

anode buffer I

Fig. 40: Diagram of a horizontal graphiteblotter for semi dry blotting. Up to sixtrans units can be blotted at one time(Kyhse-Andersen, 1984).

Graphite is the best materialfor electrodes in semi dry blot-ting because it conducts well,does not overheat and does notcatalyze oxidation products.

In semi dry blotting as well,several blots can be madesimultaneously. They are piledin layers called “trans units”.

Johansson K-E. Electrophoresis8 (1987) 379–383.

4 Blotting

4.3Blotting membranes

Nitrocellulose is the most commonly used membrane. It is available inpore sizes from 0.05 lm to 0.45 lm. The pore size is a measure ofthe specific surface: the smaller the pores, the higher the binding ca-pacity.

Proteins adsorbed on nitrocellulose can be reversibly stained sothat the total protein can be estimated before specific detection (Sali-novich and Montelaro, 1986).

It is also possible to render the blots totally transparent by animbedding technique.

Nitrocellulose is occasionally also used for preparative methodsand the proteins can be eluted out again (Montelaro, 1987).

A better adsorption of glycoproteins, lipids and carbohydrates isobtained by ligand precoating of nitrocellulose (Handmann and Jar-vis, 1985).

Polyvinylidenedifluoride (PVDF) membranes on a Teflon base pos-sess a high binding capacity and a high mechanical stability likenylon membranes. PVDF membranes can also be used for direct pro-tein sequencing (Matsudaira, 1987).

Diazobenzyloxymethyl and diazophenylthioether papers (DBM,DPT), which must be chemically activated before use, enable a twostep binding with molecules: electrostatic and covalent.

Nylon membranes possess a high mechanical stability and a highbinding capacity, usually due to electrostatic interactions. This meansthat staining can be a problem because small molecules are alsostrongly bound.

Fixing with glutardialdehyde after transfer is recommended toincrease the binding of low molecular weight peptides to nylon mem-branes (Karey and Sirbasku, 1989).

72

Fig. 41: Instrument for complete and trouble-free separation of gels backed by films.

Disadvantages: limited bindingcapacity and poor mechanicalstability.

Salinovich O, Montelaro RC.Anal Biochem. 156 (1986)341–347.

see the instructions in thesecond part.

Montelaro RC. Electrophoresis8 (1987) 432–438.

Handmann E, Jarvis HM. JImmunol Methods. 83 (1985)113–123.

Disadvantages: staining is notreversible.Matsudaira P. J Biol Chem.262 (1987) 10035–10038.

DBM and DPT are more andmore often replaced by nylonmembranes.

Positively and negativelycharged but also neutral nylonmembranes exist.

Karey KP, Sirbasku DA. AnalBiochem. 178 (1989)255–259.

4.4 Buffers for electrophoretic transfers

Ion-exchange membranes, diethylaminoethyl (DEAE) or carboxy-methyl (CM) are used for preparative purposes because of the revers-ibility of the ionic bonds.

Activated glass fiber membranes are used when blotted proteins aredirectly sequenced. Several methods to activate the surface exist: forexample, bromocyanide treatment, derivatization with positivelycharged silanes (Aebersold et al. 1986) or hydrophobation by silicona-tion (Eckerskorn et al. 1988).

Unfortunately a blotting membrane which binds 100% of the mol-ecules does not exist yet. For example, during electroblotting smallproteins often migrate through the film while larger proteins havenot yet completely left the gel. It is therefore necessary to try andobtain as regular transfers as possible.

4.4Buffers for electrophoretic transfers

A. Proteins

Tank blotting

A Tris-glycine buffer of pH 8.3 is usually used for tank blotting ofSDS gels (Towbin et al. 1979). This moderate pH should cause littledamage to the proteins. 20% methanol is often added to the buffer toincrease the binding capacity of the blotting membrane and to pre-vent the gel from swelling. At this relatively low pH value the proteinsare not highly charged so the transfers are time-consuming and thebuffer heats up gradually.

Since SDS, methanol, heat and long transfer times are also harm-ful to proteins, it can be an advantage to use buffers with higher pHvalues, this saves a considerable amount of time. For example titrate50 mmol/L CAPS to pH 9.9 with sodium hydroxide.

Acid gels with basic proteins and isoelectric focusing are blotted ina tank with 0.7% acetic acid (Towbin et al. 1979).

For glycoproteins, polysaccharides and also lipopolysaccharides 10mmol/L sodium borate pH 9.2 (Reiser and Stark, 1983) is recom-mended since boric acid binds to the sugar moieties and thus, in abasic medium, the molecules acquire a negative charge.

73

Disadvantage: Thesemembranes are often verybrittle.

Aebersold RH, Teplow D,Hood LE, Kent SBH. J BiolChem. 261 (1986) 4229–4238.

Eckerskorn C, Mewes W,Goretzki H, Lottspeich F. Eur JBiochem. 176 (1988)509–519.

Sometimes to speed up thetransfer, ready-stained proteinsfor blotting and 0.02 to 0.1%SDS to keep hydrophobicproteins in solution are addedto the buffer. But this can leadto problems with the bindingcapacity of the membrane.

This transfer takes about 50min so the buffer does not havetime to warm up.

The proteins then migratetowards the cathode.

Reiser J, Stark GR. MethodsEnzymol. 96 (1983) 205–215.

4 Blotting

Semi dry blotting

Up to now a continuous Tris-glycine-SDS buffer was often recom-mended for horizontal blotting (semidry or graphite plate blotting).Yet experience has shown that in general a discontinuous buffer sys-tem is preferable since it yields sharper bands and more regular andefficient transfers.

Continuous buffer:

48 mmol/L Tris, 39 mmol/L glycine, 0.0375% (w/v) SDS, 20% metha-nol.

Discontinuous buffer system (acc. to Kyhse-Andersen, 1984):

Anode I: 0.3 mol/L Tris, 20% methanolAnode II: 25 mmol/L Tris, 20% methanolCathode: 40 mmol/L 6-aminohexanoic acid

same as e-aminocaproic acid),20% methanol, 0.01% SDS

If the transfer efficiency of high molecular weight proteins(80 kDa) is not satisfactory, the gel can be equilibrated in the cathodebuffer for 5 to 10 min before blotting.

For enzyme detection the buffer must not contain any methanol,otherwise biological activity is lost. Brief contact with a small amountof SDS does not denature the proteins.

For transfers from urea IEF gels the urea should first be allowed toelute out of the gel by soaking it in cathode buffer. Otherwise the pro-teins, which possess no charge after IEF cannot bind to SDS as re-quired for electrophoretic transfer.

B. Nucleic acids

Tank blotting

Acid buffers are often used for DNA blotting: 19 mmol/L sodiumphosphate, 54 mmol/L sodium citrate pH 3.0 (Smith et al. 1984).

Semi-dry blotting

For neutral blotting 10 mmol/L Tris-HCl, 5 mmol/L sodium acetate,0.5 mmol/L EDTA, pH 8.7 are used. For alkaline blotting 0.4 mol/LNaOH is used (Fujimura et al. 1988). Both these techniques can becarried out in tanks with equal efficiency. However, alkaline blottingdamages the plastic material of the tank while graphite plates areresistant to sodium hydroxide.

74

A continuous buffer must ofcourse be used when it is neces-sary to carry out a doublereplica electroblotting with thesemi dry technique.

This buffer system can be usedfor SDS as well as for nativeand IEF gels.

This is mainly applicable toSDS gels.

Caution: proteins can diffuserapidly out of IEF gels.

Smith MR, Devine CS,Cohn SM, Lieberman MW.Anal Biochem. 137 (1984)120–124.

Fujimura RK, Valdivia RP, Alli-son MA. DNA Prot EngTechnol. 1 (1988) 45–60.

4.6 Blocking

4.5General staining

It is often desirable to check the electrophoresis and/or the results ofthe transfer overall before carrying out a precise evaluation.

Nucleic acids are in general visualized with ethidium bromide,which is often added to the gel before separation. The nucleic acidscan then be seen under UV light.

For proteins, besides staining with Amido Black or Coomassie Bril-liant Blue, mild staining methods such as the very sensitive IndianInk method (Hancock and Tsang, 1983) exist as well as reversibleones with Ponceau S (Salinovich and Montelaro, 1986) or Fast GreenFCF (see part II, method 9). The sensitivity of Indian Ink stainingand the antibody reactivity of the proteins can be enhanced by alka-line treatment of the blotting membrane (Sutherland and Skerritt,1986).

In many cases one can also use:

. a general immunostain (Kittler et al. 1984),

. colloidal gold (Moeremans et al. 1985),

. autoradiography,

. fluorography (Burnette, 1981).

Nylon membranes bind anionic dyes very strongly so normal stain-ing is not possible but nylon membranes can be stained with caccody-late iron colloid (FerriDye) (Moeremans et al. 1986).

4.6Blocking

Macromolecular substances which do not take part in the visualiza-tion reaction are used to block the free binding sites on the mem-brane.

Denhardts buffer is used for nucleic acids (Denhardt, 1966): 0.02%BSA, 0.02% Ficoll, 0.02% polyvinylpyrollidene, 1 mmol/L EDTA,50 mmol/L NaCl, 10 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.0, 10to 50 mg heterologous DNA per mL.

A number of possibilities exist for proteins, 2% to 10% bovinealbumin is used most often (Burnette, 1981). The cheapest blockingsubstances and the ones that cross-react the least are: skim milk or5% skim milk powder (Johnson et al. 1984), 3% fish gelatin, 0.05%Tween 20. The blocking step is quickest and most effective at 37 �C.

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Hancock K, Tsang VCW. AnalBiochem. 133 (1983)157–162.Sutherland MW, Sker-ritt JH. Electrophoresis 7(1986) 401–406.Kittler JM, Meisler NT, Viceps-Madore D. Anal Biochem. 137(1984) 210–216.Moeremans M, Daneels G,De Mey J. Anal Biochem. 145(1985) 315–321.

Moeremans M, De Raeymae-ker M, Daneels G, De Mey. J.Anal Biochem. 153 (1986)18–22.

Denhardt D. Biochem BiophysRes Commun. 20 (1966) 641–646.

Johnson DA, Gautsch JW.Sportsman JR. Gene AnalTechnol. 1 (1984) 3–8.

4 Blotting

4.7Specific detection

Hybridization

. Radioactive probes

A higher detection sensitivity can be obtained for the analysis of DNAfragments with radioactive DNA or RNA probes which bind to com-plementary DNA or RNA on the blotting membrane (Southern, 1975;Alwine et al. 1977).

. Non-radioactive probes

There is now a trend to avoid radioactivity in the laboratory, so accord-ingly the samples can be marked with biotin-streptavidin or dioxi-genin.

This method is also used for DNA fingerprinting.

Enzyme blottingThe transfer of native separated enzymes on to blotting membraneshas the advantage that the proteins are fixed without denaturationand thus do not diffuse during slow enzyme-substrate reactions andthe coupled staining reactions (Olsson et al. 1987).

ImmunoblottingSpecific binding of immunoglobulins (IgG) or monoclonal antibodiesare used to probe for individual protein zones after blocking. An addi-tional marked protein is then used to visualize the zones. Once againseveral possibilities exist:

. Radioiodinated protein A

The use of radioactive protein A which attaches itself to specific bind-ing antibodies enables high detection sensitivities (Renart et al. 1979).But, 125I-protein A only binds to particular IgG subclasses; in additionradioactive isotopes are now avoided as much as possible in the labo-ratory.

. Enzyme coupled secondary antibodies

An antibody to the specific binding antibody is used and it is conju-gated to an enzyme. Peroxidase (Taketa, 1987) or alkaline phospha-tase (Blake et al. 1984) are usually employed as the conjugatedreagent.

The ensuing enzyme-substrate reactions have a high sensitivity.The tetrazolium method has the highest sensitivity in the peroxidasemethod (Taketa, 1987).

76

Radioactive labeling is mainlyused for the evaluation of RFLPanalysis.

as for immunoblotting

Characterization of individualsin forensic medicine.

Renart J, Reiser J, Stark GR.Proc Natl Acad Sci USA. 76(1979) 3116–3120.

Taketa K. Electrophoresis 8(1987) 409–414.Blake MS, Johnston KH,Russell-Jones GJ. AnalBiochem. 136 (1984)175–179.

4.7 Specific detection

. Gold coupled secondary antibody

Detection by coupling the antibody to colloidal gold is very sensitive(Brada and Roth, 1984): in addition the sensitivity can be increased bysubsequent silver enhancement: the lower limit of detection liesaround 100 pg (Moeremans et al. 1984).

. Avidin biotin system

Another possibility is the use of an amplifying enzyme detection sys-tem. The detection results from enzymes which are part of a non-covalent network of polyvalent agents (antibodies, avidin): for exam-ple biotin-avidin-peroxidase complexes (Hsu et al. 1981) or complexeswith alkaline phosphatase.

. Chemiluminescence

The highest sensitivity without using radioactivity can be achievedwith enhanced chemiluminescent detection methods (Laing, 1986).In Fig. 42 it is shown, how such a signal can be further increased bycombining the biotin-streptavidin peroxidase system with enhancedchemiluminescence.

77

target protein

acridansubstrate

Lightemission

acridiniumesters

peroxide

primary antibody

secondaryantibody-biotinconjugate

streptavidinhorseradish peroxidasecomplex

Fig. 42: Schematic diagram of enhanced chemiluminescence plus biotin-streptavidin complexes, which afford the highest detection sensitivity.

Brada D, Roth J. Anal Biochem.142 (1984) 79–83.Moeremans M, Daneels G,Van Dijck A, Langanger G,De Mey J. J Immunol Methods.74 (1984) 353–360.

Hsu D-M, Raine L, Fanger H.J Histochem Cytochem. 29(1981) 577–580.

Laing P. J Immunol Methods.92 (1986) 161–165.

4 Blotting

To detect the signal, the membrane is exposed to a film or to anappropriate CCD camera in an absolutely dark closet for a certaintime period. New multipurpose scanners can also be used in a chemi-luminescence detection mode.

Immunological detection on blots can be automated, for examplewith the staining unit of the PhastSystem� (Prieur and Russo-Marie,1988).

Lectin blottingThe detection of glycoproteins and specific carbohydrate moieties canbe performed with lectins. Visualization is carried out by aldehydedetection or, analogous to immunoblotting, with the avidin-biotinmethod (Bayer et al. 1987).

4.8Protein sequencing

The use of blotting for direct protein sequencing has been a big stepforward for protein chemistry and molecular biology (Vande-kerckhove et al. 1985). Blotting is mostly performed out of one-dimensional SDS gels or 2D gels (Matsudaira et al. 1987; Aebersold etal. 1986; Eckerskorn et al. 1988; Eckerskorn and Lottspeich, 1989). Ifthe proteins to be sequenced have to be separated by isoelectric focus-ing, an immobilized pH gradient should be used because carrierampholytes would interfere with the sequencing signals (Aebersold etal. 1988).

Also matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS) has been employed to measure the molecular mass ofproteins from blots with high precision (Eckerskorn et al. 1992; Stru-pat et al. 1994).

The reference by Simpson et al. (1989) presents a review of thedifferent methods.

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Prieur B, Russo-Marie F. AnalBiochem. 172 (1988) 338–343.

Bayer EA, Ben-Hur H,Wilchek M. Anal Biochem. 161(1987) 123–131.

Vandekerckhove J, Bauw G,Puype M, Van Damme J, VanMontegu M. Eur J Biochem.152 (1985) 9–19.Eckerskorn C, Lottspeich F.Chromatographia. 28 (1989),92–94.Aebersold RH, Pipes G,Hood LH, Kent SBH. Electro-phoresis 9 (1988) 520–530.Eckerskorn C, Strupat K,Karas M, Hillenkamp F,Lottspeich F. Electrophoresis 13(1992) 664–665.Strupat K, Karas M,Hillenkamp F, Eckerskorn C,Lottspeich F. Anal Chem. 66(1994) 464–470.

Simpson RJ, Moritz RL,Begg GS, Rubira MR, Nice EC.Anal Biochem. 177 (1989)221–236.

4.9 Transfer problems

4.9Transfer problems

. Poor solubility, especially of hydrophobic proteins, can preventa transfer. In such cases the blotting buffer should contain adetergent, for example SDS. The addition of urea can alsoincrease the solubility.

. During native immunoblotting according to Bjerrum et al.(1987), the electrophoresis gel contains non-ionic detergents.Its binding capacity is hindered in case of direct contact withthe blotting membrane. This can be prevented by inserting a 2to 3 mm thick agarose gel layer containing transfer buffer butno detergent between the gel and the blotting membrane.Nitrocellulose with increased binding capacity can also beused.

. High molecular weights cause a slower migration out of thegel. But when blotting is carried out for a long time and/or athigh field strengths, low molecular weight proteins detachthemselves again from the membrane and are lost. Severalpossibilities to obtain regular transfers over a wide molecularweight spectrum exist:

. The use of pore gradients in SDS-PAGE,

. The use of a discontinuous buffer system for semi dry blot-ting,

. The treatment of high molecular weight proteins with proteaseafter electrophoresis,

. The use of a buffer with another pH,

. The use of a buffer without methanol,

. The addition of SDS (0.01 to 0.1%) to the buffer,

. The use of native immunoblotting (Bjerrum et al. 1987),

. Blotting for a longer time and placing a second blotting mem-brane behind.

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The addition of 6 to 8 mol/Lurea is only reasonable in semidry blotting (small buffervolume).

Bjerrum OJ, Selmer JC,Lihme A. Electrophoresis 8(1987) 388–397.

After the separation theproteins are distributedaccording to their molecularweight in areas with small orlarge pores.

The isotachophoresis effectinduces a regular velocity.

Limited proteolysis usually doesnot damage the antigenicity.

This increases the mobility.

The pores become larger whenthe gel swells.

Caution: too much SDSreduces the binding capacity.

Gels with large pores can beused here,

To trap low molecular weightproteins.

4 Blotting

Applications of protein blottingA series of review articles has been published:Gershoni JM, Palade GE. Anal Biochem. 112 (1983) 1–15.Beisiegel U. Electrophoresis 7 (1986) 1–18.Bjerrum OJ. Ed. Paper symposium protein blotting. Electrophoresis 8 (1987)

377–464.Baldo BA, Tovey ER. Ed. Protein blotting. Methodology, research and diagnostic

applications. Karger, Basel (1989).Baldo BA. In Chrambach A, Dunn M, Radola BJ. Eds. Advances in electrophore-

sis 7. VCH, Weinheim (1994) 409–478.

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