Protein blotting a review(1).pdf

ReviewProtein blotting:a reviewBiji T.Kuriena,*,R.Hal Scofielda,b,caArthritis and Immunology Program,Oklahoma Medical Research Foundation,825 NE 13th Street,Oklahoma City,OK 73104,USAbDepartment of Medicine,University of Oklahoma Health Sciences Center,Oklahoma City,OK 73104,USAcDepartment of Veterans Affairs Medical Center,Oklahoma City,OK 73104,USAReceived 26 August 2002;received in revised form 15 November 2002;accepted 22 November 2002Keywords:Western blotting;SDS-PAGE;Nitrocellulose1.IntroductionThe utility of the high resolving power of gelelectrophoresis was limited in purpose,owing to thefact that the separated proteins in the gel matrix weredifficult to access with molecular probes,until theadvent of a procedure that allowed transfer of proteinsfrom the gel to an adsorbent membrane(Towbin et al.,1979).Protein blotting evolved as an offshoot of DNA(Southern)blotting(Southern,1975)and RNA(North-ern)blotting(Alwine and Kemp,1977).As an effort toretain the geographic naming tradition initiated bySouthern(1975),Burnette(1981)coined the termWestern blotting to describe a slightly modifiedprocedure from that of Towbin et al.(1979).Westerntransfer with subsequent immunodetection is a power-ful tool to detect and characterize a multitude ofproteins,especially those proteins that are of lowabundance.It involves the transfer of proteins sepa-rated on sodium dodecyl sulfate polyacrylamide gelelectrophoresis(SDS-PAGE)(Laemmli,1970)to asolid support.The blotted proteins form an exactreplica of the gel and have proved to be the startingstep for a variety of experiments.The subsequentemployment of antibody probes directed against thenitrocellulose bound proteins has revolutionized thefield of immunology.Since its inception,protein blot-ting has been evolving constantly and now the scien-tific community is confronted with a plethora of waysand means of transferring proteins.1.1.Efficiency of protein blottingTwo major factors affect the efficiency of proteinblotting:(1)the elution efficiency of a protein out of agel matrix and(2)the efficiency of binding by themembrane.1.1.1.The elution efficiency of a protein out of a gelmatrixAll blotting techniques require a successful transferof proteins from the gel to a solid membrane support.Therefore,some consideration should be given to thenature of the gel used.In a general way,the lower thepercentage of acrylamide and cross-linker,the easierthe transfer will be.Running the softest gel that yieldsthe required resolution is the best option.Transferbecomes faster and more complete with the use ofthinner gels.Use of ultra-thin gels,however,maycause handling problems and a 0.4-mm thickness0022-1759/02/$-see front matter D 2002 Elsevier Science B.V.All rights reserved.doi:10.1016/S0022-1759(02)00523-9*Corresponding author.Tel.:+1-405-271-7394;fax:+1-405-271-7063.E-mail address:biji-kurienomrf.ouhsc.edu(B.T.Kurien) of Immunological Methods 274(2003)115represents the lower practical limit(Harlow and Lane,1988).High-molecular-weight proteins blot poorly fol-lowing SDS-PAGE,which results in low levels ofdetection on immunoblots.Therefore,investigatorsanalyzing large proteins have sought to increase theefficiency of transfer by enhancing the degree ofprotein migration out of the gel during the transferby methods ranging from disruption of the gel matrixto partial proteolytic digestion of the proteins prior tothe transfer(Renart et al.,1979;Elkon et al.,1984;Gibson,1981).Recently,some investigators haveshown very efficient,rapid and convincing methodsto transfer high-molecular-weight proteins onto mem-branes(Bolt and Mahoney,1997;Kurien and Sco-field,2002).1.1.2.Efficiency of binding to the membrane(Harlowand Lane,1988)Nitrocellulose,polyvinylidene difluoride(PVDF),activated paper or activated nylon have all been usedsuccessfully to bind transferred proteins(Gershoniand Palade,1983;Renart et al.,1979;Towbin et al.,1979).The most commonly used membrane support,nitrocellulose,is disadvantageous in the fact that theproteins are not covalently bound and it is brittle whendry.Small proteins tend to move through nitrocellu-lose membranes and only a small fraction of the totalamount actually binds.This can be obviated by usingmembranes with smaller pores.Some investigatorshave used gelatin-coated nitrocellulose for quantita-tive retention(Too et al.,1994).In supported nitro-cellulose(e.g.,Hybond-C Extra),the mechanicalstrength of the membrane has been shown to beimproved by incorporating a polyester support web,thereby making handling easier.PVDF membranes offer the advantages of highprotein binding capacity,physical strength and chem-ical stability.One of the advantages of electroblottingproteins onto PVDF membranes is that replicate lanesfrom a single gel can be used for various purposessuch as N-terminal sequencing,proteolysis/peptideseparation/internal sequencing and other analysismethods such as Western analysis.In addition,whilenitrocellulose membranes cannot be stained withCoomassie Brilliant Blue(CBB),PVDF membranesare amenable to staining with CBB,thus allowingexcision of proteins for N-terminal sequencing.Activated paper(diazo groups)binds proteinscovalently,but finds its main drawback in that thecoupling method is incompatible with many gelelectrophoresis systems.Linkage is through primaryamines and therefore systems that use gel bufferswithout free amino groups must be used with thispaper.In addition,the paper is expensive and thereactive groups have a limited half-life once the paperis activated.Nylon has excellent mechanical strengthbut can only bind a small amount of protein and is notsuitable for most applications.Some of these prob-lems are solved by using activated nylon(positivelycharged)which,however,produces higher non-spe-cific binding.On account of all these problems,nitrocellulose membranes have remained the bestcompromise for most situations.2.Methods to transfer proteins from gel tomembraneTransfer of proteins from native or SDS-PAGE gelsto nitrocellulose or PVDF membranes has beenachieved in three different ways:(1)simple diffusion(Renart et al.,1979;Kurien and Scofield,1997);(2)vacuum-assisted solvent flow(Peferoen et al.,1982);and(3)Western blotting or electrophoretic elution(Towbin et al.,1979).Comprehensive reviews have been undertaken byseveral investigators(Gershoni and Palade,1983;Towbin et al.,1989;Harper et al.,1990;Egger andBienz,1994;Wisdom,1994).A multitude of papershave been published since then,entailing modifiedversions of the original protocol.We prefer to classifythe various methods based on the three differentmeans of transferring proteins to membranes:(1)simple diffusion,(2)vacuum-assisted solvent flowand(3)Western blotting or electrophoretic elution.Finally,a brief description of methods generally usedto detect antigens on blots will be provided.2.1.Simple diffusionDiffusion blotting was originally developed as ameans of transferring proteins separated by iso-elec-tric focusing on thin gels to membranes and then laterexpanded to other gel systems(Reinhart and Mala-mud,1982;Jagersten et al.,1988;De Keyser et al.,B.T.Kurien,R.H.Scofield/Journal of Immunological Methods 274(2003)11521990;Heukeshoven and Dernick,1995;Kurien andScofield,1997;Olsen and Wiker,1998;Chen andChang,2001).This procedure requires the laying of amembrane on the surface of the gel with a stack of dryfilter paper on top of the membrane.A glass plate andan object with a certain weight are usually placed onthis assembly to facilitate the diffusion process.Kur-ien and Scofield(1997)resuscitated a waning interestin diffusion transfer by sandwiching the gel betweentwo membranes supported by filter paper and glassplates on either side to obtain two blots.They furtherrepeated the procedure to obtain a total of 12 blotsfrom a single gel.Renart et al.(1979)and Bowen et al.(1980)hadshown the simple diffusion of proteins to membranes.However,this protocol had not gained widespreadacceptance owing to the fact that there was noquantitative transfer of protein.Since the demonstra-tion that multiple immunoblots can be generated afternon-electrophoretic bidirectional transfer of a singleSDS-PAGE gel with multiple antigens,there is con-siderable interest in diffusion-mediated transfer ofproteins(Kurien and Scofield,1997).This protocolinvolves incubating the membrane on either side of agel at 37 jC for 2,4 or 10 h.Twelve blots can beobtained using this procedure.Lifts from SDS-PAGEgels for immunoblotting using this method are partic-ularly useful in identification of proteins by massspectrometry(Kurien and Scofield,2000,2001).Afterdiffusion blotting the gel was stained with CBBand the antigens on the blot were detected by immu-nostaining.The immunoblotted target band wascompared with the Coomassie-stained gel by super-imposing the blot and the stained gel,allowing theidentification of the band to be excised for trypticdigestion for subsequent mass spectrometric analysis.The greatest advantage of diffusion blotting comparedto electroblotting is that several transfers or imprintscan be obtained from the same gel and differentantisera can be tested on identical imprints.Subsequently,Olsen and Wiker(1998),followedby Chen and Chang(2001),have corroborated theusefulness of the diffusion transfer process.Theformer have demonstrated the efficiency of proteintransfer using14C labeled proteins.A 3-min diffusionblotting was shown to give a transfer of 10%compared to electroblotting.They were able to obtainat least 10 imprints,from the same gel,with all theproteins visible in all the imprints without anydetectable loss in resolution as compared to electro-blotting.Zymography,or activity gel electrophoresis,hasalso been studied in regards to the utility of diffusion.Zymography is a technique that involves the electro-phoresis of enzymes(either nucleases or proteases)through discontinuous polyacrylamide gels containingenzyme substrate(either type III gelatin or B-casein).After electrophoresis,SDS is removed from the gel bywashing in 2.5%Triton X-100 solution.This allowsthe enzyme to renature,and the substrate is degraded.Staining of the gel with CBB(in the case of proteins)allows the bands of enzyme activity to be detected asclear bands of lysis against a blue background(Bis-choff et al.,1998).In this procedure,an additionalimmunoblotting analysis using another gel is oftenrequired to examine a particular band that is involved.Chen and Chang(2001)have used diffusion blottingto circumvent the use of second gel for this purpose.They blotted the activity gel onto PVDF for immu-nostaining and the remaining gel after blotting wasused for routine activity staining.Because the blotand the activity staining are derived from the samegel,the signal localization in the gel and the replicacan be easily aligned for comparison.Chen and Chang have shown that diffusion blot-ting transfers 2550%of the proteins to the mem-brane compared to electroblotting.However,theadvantage of obtaining multiple blots from the samegel could outweigh the loss in transfer and actuallycould be compensated for by using sensitive detectiontechniques.The gel remains on its plastic support,which prevents stretching and compression;thisensures identical imprints and facilitates more reliablemolecular mass determination.If only a few imprintsare made,sufficient protein remains within the gel forgeneral protein staining.These advantages make dif-fusion blotting the method of choice when quantita-tive protein transfer is not required.There are a number of advantages to this proce-dure over the conventional upright,electrophoreticprotocol,not the least of which is that as little as acouple of hundred millilitres of buffer is all that isneeded for electroblotting several gels,compared toas much as 5 l for some commercial kits.In addition,as noted above,several gels can be blotted simulta-neously.B.T.Kurien,R.H.Scofield/Journal of Immunological Methods 274(2003)11532.2.Vacuum blottingPeferoen et al.(1982)developed a simple blottingmethod as an alternative to diffusion blotting andelectroblotting.They used the suction power of apump connected to a slab gel dryer system to drivethe separated polypeptides from the gel to the nitro-cellulose membrane.Both low-and high-molecular-weight proteins could be transferred using thismethod.When using low-molecular-weight proteins,the authors suggest using nitrocellulose membranewith a small pore size(0.2 or 0.1 Am),since small-molecular-weight proteins(F14,000 Da)are lessadsorbed by the 0.45-Am membrane.There is the possibility of the gel drying if theprocedure is carried out over longer periods(45 min)and enough buffer should be used in such cases.Insome cases low concentration polyacrylamide gelsstuck to the membrane after the transfer.In suchcases,the authors suggest rehydrating the gel,whichmakes it swollen,and then nitrocellulose membranecan be easily detached from the gel remnants.2.3.ElectroblottingElectroblotting has been and is the most commonlyused method to transfer proteins from a gel to amembrane.The principal advantages are the speedand the completeness of transfer compared to diffusionor vacuum blotting.Electroelution can be achievedeither by(1)complete immersion of a gel-membranesandwich in a buffer(wet transfer)or by(2)placing thegel-membrane sandwich between absorbent papersoaked in transfer buffer(semi-dry transfer).2.3.1.Wet transferFor the wet transfer,the sandwich is placed in abuffer tank with platinum wire electrodes(Fig.1).There are a large number of different apparatuses thatwill efficiently transfer proteins(or other macromole-cules)transversely from gel to membrane.Most ofthese,however,are based on the design of Towbin etal.(1979):i.e.,they have vertical stainless steel/platinum electrodes in a large tank.2.3.2.Semi-dry transferFor the semi-dry transfer,the gel-membrane sand-wich is placed between carbon plate electrodes.Semi-dry or horizontal blotting uses two plateelectrodes(stainless steel or graphite/carbon)for uni-form electrical field over a short distance,and sand-wiches between these up to six gel/membrane/filterpaper assemblies,all well soaked in transfer buffer.The assembly is clamped or otherwise secured on itsside,and electrophoretic transfer effected in thisposition,using as transfer buffer only the liquidcontained in the gel and filter papers or other padsin the assembly.There are a number of advantages to this procedureover the conventional upright protocol.Several gelscan be blotted simultaneously;electrodes can becheap carbon blocks;less power is required for trans-fer(and therefore a simpler power pack).3.Modifications of electroblottingFrom the basic electroblotting procedure,severalprotocols have been developed.Among these are anumber of attempts to improve the original protocol,focusing on increasing the amount of protein trans-ferred and retained on the membrane.Fig.1.Western blot transfer sandwich assembly for wet transfer.The transfer membrane is sandwiched between the