Journal of Pharmacological and Toxicological Methods 61 (2010) 171–177

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Original article

Methodological considerations for improving Western blot analysis

Daniel J. MacPhee ⁎Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada A1B 3V6

⁎ Division of BioMedical Sciences, Health Sciences CPhilip Drive, St. John's, NL, A1B 3V6. Tel.: +1 709 777 8

E-mail address: dmacphee@mun.ca.

1056-8719/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.vascn.2009.12.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 November 2009Accepted 7 December 2009

Keywords:ImmunoblottingMethodsOptimizationProtein blottingStainsWestern blot

The need for a technique that could allow the determination of antigen specificity of antisera led to thedevelopment of a method that allowed the production of a replica of proteins, which had been separatedelectrophoretically on polyacrylamide gels, on to a nitrocellulose membrane. This method was coinedWestern blotting and is very useful to study the presence, relative abundance, relative molecular mass, post-translational modification, and interaction of specific proteins. As a result it is utilized routinely in manyfields of scientific research such as chemistry, biology and biomedical sciences. This review serves to touchon some of the methodological conditions that should be considered to improve Western blot analysis,particularly as a guide for graduate students but also scientists who wish to continue adapting this nowfundamental research tool.

entre, Rm 5340B, 300 Prince768; fax: +1 709 777 7010.

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© 2009 Elsevier Inc. All rights reserved.

1. Introduction

Theneed to solve a problem leads to innovation and in the late 1970stherewas a real need for a technique that could allow the determinationof antigen specificity of antisera. This led to the development of amethod that allowed the production of a replica of proteins, which hadbeen separated electrophoretically on polyacrylamide gels, on to anitrocellulose membrane (Fig. 1). This replica could subsequently beprobed with antisera to detect specific proteins. Such a method wasindependently developed by two laboratories, onemethod for use withpolyacrylamide–urea gels (Towbin, Staehelin, & Gordon, 1979; Towbin,2009) the other for the more widely employed sodium dodecyl sulfate(SDS)–polyacrylamide gels (Burnette, 2009; Burnette, 1981). The lattermethod was coined Western blotting (Burnette, 1981) although termssuch as protein blotting and immunoblotting can also be used.Thereafter, it quickly became recognized as a powerful tool to studythe presence, relative abundance, relative molecular mass, post-translational modification, and in partnership with immunoprecipita-tion the specific interaction of proteins (Kurien& Scofield, 2009a,b). As aresult it is utilized in many facets of life science research — a rathertremendous achievement given that one of the research papersdescribing the techniquewas rejected outright at first (Burnette, 2009).

As proof of the usefulness ofWestern blotting, a term that continueda directional theme following Northern blotting and based on the use ofmembranes for nucleic acid blotting by Southern (Alwine, Kemp, &Stark, 1977; Southern, 1975), the technique has continued to evolve andvast information exists on troubleshooting and improving the sensitiv-

ity, speed, and quantitation of the technique (for a recent thoroughtreatment see Kurien & Scofield, 2009a,b). This review serves to touchon some of the methodological conditions that should be considered toimprove Western blot analysis.

2. Attend to lysis conditions

For many, Western blotting begins with the running of a polyacryl-amide gel containing a mixture of proteins for subsequent blotting.However, it is important to consider the quality of the protein lysateproduced at the outset. There exists a vast array of different tissue/celllysis buffers (Harlow & Lane, 1999) but one must consider if a particularbuffer is optimally designed to extract the proteins of interest,particularly the use of the appropriate detergent for protein extractionand solubilization. For example, integrin receptors are integral mem-brane proteins and while buffers such as Nonidet P-40 (NP-40) andradioimmunoprecipitation assay (RIPA) lysis buffers extract manyproteins (Harlow & Lane, 1999), the use of the detergent octylglucosideis a far better choice to obtain a high yield of such receptors compared touse of NP-40 or Triton X-100 detergents for example (Elbe &Berditchevski, 2002). This would be particularly important if relativeabundance of these proteins was going to be calculated between controland experimental conditions. Of note, the detergent utilizedmust still becompatible for subsequent polyacrylamide gel electrophoresis. For moredetailed information on the use of different detergents see Helenius andSimons (1975) and Helenius, McCaslin, Fries, and Tanford (1979). Thus,the key features a lysis buffer must possess for subsequent proteinextraction, polyacrylamide gel electrophoresis, andWestern blotting areefficient protein extraction ability and the capability tomaintain antiserarecognition of the protein (antigenic sites).

Fig. 1. A schematic of the electroblot transfer process using an immersion procedure. Arrow at the bottom of the tank indicates transfer direction.

172 D.J. MacPhee / Journal of Pharmacological and Toxicological Methods 61 (2010) 171–177

If co-precipitating proteins were sought after in the subsequentWestern blotting procedure, then a lysis buffer that extracted theproteins sufficiently while maintaining protein–protein interactionswould be preferable (Harlow & Lane, 1999). For immunoprecipitation,the cells or tissues should be lysed utilizing a detergent, such as a non-ionic detergent (e.g. NP-40), that is gentle enough to maintain theconformation or enzymatic activity of the target protein(s) but stillcapable of breaking up cellular membranes, weak protein–proteininteractions, and result in release of most proteins from the cells ortissue(s). There are situations however, where more harsh extractionconditions (e.g. SDS or deoxycholate) are required. These specificrequirements for protein extractions should thus be considered prior toconducting immunoprecipitations. In total, the utility and effectivenessof the Western blot analysis will only be as good as the protein lysatesprepared for polyacrylamide gel electrophoresis (PAGE).

If archiving your samples, cells and tissues should be frozen rapidlyfor storagewith liquidnitrogen to avoidproteasedegradationof proteinsin the sample or collected and lysed quickly, preferably while chilled.Sinceproteases canbe releasedduring lysis, aswell as phosphatases, andact on your target protein(s), protease and phosphatase inhibitorsshould be included in the lysis buffers. Many of these are produced ascocktails in tablet form for easy purchase and their use is as simple asdissolving the tablet in the lysis buffer prior to use (e.g. Complete Mini,Protease Inhibitor Cocktail Tablets, PhosSTOP tablets, Roche Diagnos-tics). While there are many different lysis buffers, my laboratory likes touse a variation of RIPA lysis buffer [50 mM Tris–HCl (pH7.5), 150 mMNaCl, 1% (vol/vol) Triton X-100, 1% (wt/vol) sodium deoxycholate, 0.1%(wt/vol) SDS] containing 100 μM Na2VO3 and COMPLETE, Mini EDTA-free protease inhibitors (Roche Molecular Biochemicals, Laval, Quebec,Canada). It contains two ionic detergents, SDS and deoxycholate, aswellas a non-ionic detergent Triton X-100 (Williams, White, & MacPhee,2005), thus it is capable of denaturing proteins, breakingmany protein–protein interactions and releasingmost cellular proteins (Harlow&Lane,1999). Another buffer, perhaps more commonly used is NP-40 lysisbuffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40) whichcontains only a non-ionic detergent and is more gentle than RIPA buffer,but still capable of solubilizing themajority of common proteins studied(Butler, Elustondo, Hannigan, & MacPhee, 2009; Harlow & Lane, 1999).Some researchers like to modify or develop their own lysis buffers inwhich case, Harlow and Lane (1999) have recommended consideringthe following range of variables to optimize the lysis buffer for Westernblot analyses: salt concentrations 0–1 M, non-ionic detergents 0.1–2%,ionic detergents 0.01–0.5%, divalent cation concentrations 0–10 mM,EDTA concentrations 0–5 mM, and pH 6–9.

3. SDS-PAGE and transfer of proteins

Since Tris–glycine SDS-PAGE is the most common strategy to elec-trophoretically separate protein forWestern blot analysis, it will be dealt

with here exclusively. The SDS-PAGE procedure has been documented indetail elsewhere (Burnette, 1981; Sambrook & Russell, 2001). Theprocedure utilizes the characteristics of SDS, a strongly anionic detergent,with or without a reducing agent and heat to dissociate proteins.Denaturedproteinsbind toSDSwithin their hydrophobic regions andasaresult become negatively charged. The key aspect of the procedure is thatthe amount of SDS that binds to the denatured proteins is approximatelyproportional to the molecular mass of the polypeptides facilitatingdirectional migration of the highly negatively charged SDS–proteincomplexes in the polyacrylamide gel based on polypeptide size.

Prior to preparation of protein samples for electrophoresis it isimportant to determine the optimal bisacrylamide:acrylamide ratio foroptimal rangeof separation and sievingproperties (Sambrook&Russell,2001). Forproteins theusual bisacrylamide–acrylamide ratio is 1:29andthe easiest and most consistent way to use the reagent is to buy itpremade and pre-mixed in lots from reputable companies. In manycases SDS-PAGE is still conducted with discontinuous buffer systems,the pH and ionic strength of the running buffer in the reservoir and ofthe buffers used to cast the gels becomes important as it has a bearingonthe resolution of the SDS-PAGE (Sambrook & Russell, 2001). Thus, sincemany of the buffers are made as stocks and kept on lab benches or infridges, they should be periodically checked for proper pH and if left forextended periods, be replaced. When the buffers are made, Tris base isrecommended to avoid production of diffuse protein bands (Sambrook& Russell, 2001).

Once electrophoresis is complete, a replica of the separated proteinscan be made on to a membrane— aWestern blot. Electroblotting is themost popular method for creating the replica, although other methodsmay have specific advantages (see below). Whether an immersion orsemi-dry electroblotting method is utilized for preparing the replica,certain common factors must be considered (Bjerrum & Schafer-Nielson, 1986; Towbin et al., 1979) such as avoiding air bubbles in thetransfer sandwiches. The use of semi-dry methods have constantlyimproved and are rapid compared to immersion, but high molecularmass proteins may not transfer as effectively as in immersion methods.There are a number of types of membranes that have been utilized overthe years, but the two that have gained the most favour arepolyvinylidene fluoride (PVDF) and nitrocellulose (Kurien & Scofield,2009a,b). These supports work by binding the proteins non-covalentlythrough primarily hydrophobic interactions (Tonkinson & Stillman,2002). Additional details about these membranes and others used inWestern blotting are provided in detail elsewhere (Kurien & Scofield,2009a,b). There are modifications to these standard membranesavailable for use, such as low fluorescence background membranes(e.g. Hybond LFP, GE Healthcare; FluorTrans, Pall Corporation; Immo-bilon-FL, Millipore Corporation) for use with direct or indirectfluorescence detection of proteins, and various nitrocellulose andPVDF membranes designed to be more efficient for binding smallermolecular mass proteins and more durable for re-probing and ECL

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detection (e.g. BioTrace NT, Pall Corporation; Immobilon P, MilliporeCorporation; Hybond P, GEHealthcare). However, in our experiences allsuppliers do not produce and/or sell membranes of equal quality andeffectiveness. Care must be taken by the laboratory to optimize themembrane type, porosity, and supplier for the specific Western blotanalyses required.

Overall protein transfer effectiveness is dependent on the type of gel(percentage acrylamide, bisacrylamide:acrylamide ratio), themolecularmass of the proteins to be transferred, and themembrane to be used forproductionof the replica. Every typeofmembranehas a limited capacityto bind protein (e.g. NC: 80–250 ug/cm2; PVDF: ∼170 ug/cm2,Sambrook & Russell, 2001) so it is prudent to consider how muchprotein lysate to run per lane on the gel. Excess protein will not bindproperly and can be lost during washes, particularly with use of non-ionic detergents. Karey and Sirbasku (1989) have reported on the use ofglutaraldehyde to fix protein to nitrocellulose and reduce protein loss.Pore size must also be considered for membranes. A 0.2 um pore sizemembrane adsorbs smaller molecular mass proteins better than a0.45 um pore membrane (Burnette, 1981). Still, numerous methodshave been developed to improve blotting of high molecular massproteins (Bolt &Mahoney, 1997;Gibson, 1981; Kurien& Scofield, 2002).In particular, Kurien and Scofield (2002) have reported an effective andrapid method for transferring proteins, particularly of high molecularmass, by using heated buffer for electroblotting. The simple techniqueutilizes a transfer buffer of 25 mM Tris and 190 mM glycine but heatedto 70–75 °C. With this method, it was reported that transfer took 5–20 min at 40 V depending on the type (percentage acrylamide) and thethickness of the polyacrylamide gel (0.75 mm or 1.5 mm). Theunderlying mechanism for this improved method is thought to beheat- induced increased permeability of the polyacrylamide gelfacilitating quick transfer of the proteins. A similar strategy has alsobeen shown to be useful for DNA elution from agarose gels (Kurien,Kaufman, Harley, & Scofield, 2001, Kurien,Matsumoto, & Scofield, 2001;Kurien & Scofield, 2006).

Another transfermethodworth considering in certain circumstancesis diffusional transfer (Kurien & Scofield, 2006). Using the simplediffusion method up to 12 blots from a single gel were reportedlyproduced by sandwiching the gel between membranes in a sequentialmanner (Kurien & Scofield, 1997). Since many gel replicas can beproduced and the gel can still retain protein, this might be quite usefulfor mass spectrometry analysis as the gel could be stained, while theblots could be probed with various antisera to identify protein(s) thusaiding the subsequent removal of several identified bands on the gel atonce (Kurien et al., 2000, 2001) increasing throughput of the assay. Thetransfer method has also proven to be useful for zymography withreplicas made for Western blot analysis of the specific enzymes ofinterest and the gel retained for development of enzymatic activity(Chen et al., 2001).

Finally, for electroblotting the researcher must decide whether ornot to utilize methanol in the transfer buffer as it is known to improvethe adsorption of protein to the membrane support, prevent gelswelling, and protein distortion during electroblotting (Towbin &Gordon, 1984). However, the use of methanol in the transfer buffercan shrink gels whichmay be problematic for electroblotting of highermolecular weight proteins. Overall, there are many variations on thetheme of electroblotting for specific purposes such as double blotting,grid immunoblotting and tissue printing. The reader is directed toconsult Kurien and Scofield (2006) for additional details.

Once a blot is produced our experience has taught us that it is stillprudent to check the effectiveness and homogeneity of the transferacross the membrane. It is also an opportunity to confirm thecomparable protein loading per gel lane that was calculated initiallywith a protein assay (e.g. Bradford assay). Both of these considerationscan be accomplished in a relatively straightforward and rapid mannerwith kits (e.g. R-PROB, Sigma-Aldrich; MemCode™, ThermoFisher;Antharavally, Carter, Bell, & Mallia, 2004) that allow reversible staining

and imaging of the newly made blot prior to the Western blot analysesand yet are compatible with subsequent immunodetection methods.The Memcode™ anionic stain produces a bluish band that is photo-graphable and the detection limit approaches 25 ng. It is also compatiblefor PVDF and nitrocellulosemembranes and both ECL and chromogenicdetectionmethods (Antharavally et al., 2004). If an individual wishes todo it themselves, then a stock solution of Ponceau S stain can be madeconsisting of 2% Ponceau S in 30% trichloroacetic acid, 30% sulfosalicyclicacid and, following staining in a 1:10 working solution, the blotscan be destained in several changes of PBS (D'Souza & Scofield, 2009;Salinovich & Montelaro, 1986; Sambrook & Russell, 2001). Of note,Ponceau S staining of blots can sometimes produce high background inthe subsequent blot development protocol. In addition, generally thesensitivity of reversible staining is not as great as with the use ofirreversible stains such as Amido Black; however, reversible staining ofthe blot will provide the user with confidence that the transfer wasuniform across the membrane— a characteristic that is essential forfuturemeasurementof relativequantities of a specific proteinof interestin the lanes of the blot. One caveat that we have encountered so far isthat destaining of blots with such systems appears, at least currently, toimpair analysis of phosphorylated proteins. This may be due to thedestain containing a low concentration of HCl. Another consideration isto use a fluorescence-based method for detecting the proteins on themembrane such as SYPRO Ruby. Berggren et al. (1999) have reportedthat use of this dye allows gentle, sensitive detection that is compatiblewith subsequent ECL detection systems inWestern blot analysis. Lastly,Yonan, Duong, and Chang (2005) have recently reported that ReactiveBrown 10, a reactive fabric dye, can rapidly and reversibly stain blotswith a sensitivity that approaches 1–2 ng. The authors utilized a 0.05%(w/v) solution of the dye in distilled water and only incubated blots for5–10 s followed by destaining inwater. To completely remove the stain,blots could be destained in 0.1 N NaOH for 10 min and the blots werestill reportedly viable for subsequent immunodetection. Certainly newreversible and sensitive stains are constantly being explored and thesewill continue to improve the rapidity and efficiency of future analyses(e.g. Pal, Godbole, & Sharma, 2004). Formore details on stains and shortprotocols for use, the reader is directed to D'Souza and Scofield (2009).

Another strategy that could be utilized is to flank the samples on thegel with pre-stained molecular mass markers and evaluate thecompleteness of their transfer on the blot. The disadvantage is that theresearcher would not be evaluating the transfer effectiveness of all lanesandwehaveencounteredonoccasion, that despite our best efforts, somelanes do not transfer as effectively as others usually due to the presenceof some small bubbles. Evaluating the transfer with a reversible stainwouldmore effectively prevent theuse of an inappropriateWestern blotfor subsequent immunodetection.

Following evaluation of the protein transfer, we also find that thisis an opportune time to consider cutting the blots with a razor blade,prior to destaining, using the pre-stainedmolecular mass standards asa guide. This will aid Western blot analysis of two to several proteinsof varying molecular mass simultaneously from the original blotsaving time and possibly precious protein samples. It may also allowthe user to cut the blot and remove unwanted portions to minimizethe size and subsequent volumes of buffers, antisera solutions, etc andreduce the cost associated with developing the blot. This would beparticularly useful when using large format polyacrylamide gels.

4. Blocking and antisera

With blots prepared, the next consideration is how to block them,particularly PVDF and nitrocellulose membranes. Usually some mixtureof proteins in a buffer is used such as 5% non-fat milk powder in Trisbuffered saline containing 0.1% Tween-20 (TBST) or 5% BSA in a similarbuffer. Iwould advise checking the specific antiseradatasheets, however,as some specifically state the use of BSA instead of milk powdercontaining blocking solutions and certain membrane supports work

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better with one blocking solution compared to another (i.e. BSA mayblock PVDFmembranes inadequately). As a result, the researcher shouldmatch the type ofmembranewith the blockingbuffer that is appropriatefor the specific antiserum. Blocking a blot serves two important pur-poses. Thefirst iswell known, in that, it can helpmask anypotential non-specific binding sites on themembrane itself. The second purpose is lessknown andperhaps even less understood, but blocking amembrane canpromote renaturation of antigenic sites (Towbin & Gordon, 1984).However, it has been reported that prolonged blocking times (N24 h)can actually remove antigens (DenHollander & Befus, 1989).

Despite the advances and newmethodologies emerging for Westernblot signal detection (see Kurien & Scofield, 2009ab) antisera are still acritical part of the procedure as the titer of the antisera is a primedeterminant of the sensitivity of the assay (Burnette, 1981). Subsequent-ly, a key optimization involves pre-determining the optimal concentra-tions of both primary and secondary antisera for analysis. The dilution ofthe antisera to be used will be determined by the avidity of the antiserautilized. Depending on the detection method used (e.g. enhancedchemiluminescence) the dilution should be optimized to ultimatelyprovide the best signal to noise ratio. This process will also be influencedby the method and sensitivity of signal detection utilized such as ECL-rated film or CCD camera systems. One quick and easy way to optimizeboth antigen and antibody concentrations prior to the actual Westernblot analysis is to conduct adot blot analysis. It is very similar to aWesternblot analysis procedure, but in this analysis different concentrations ofproteins can be spotted on membrane strips and dried for subsequentanalysis with different concentrations of primary and secondary (e.g.HRP-labeled) antisera to determine the optimal conditions (best signal tonoise ratio). The procedure also offers an opportunity to test differentblocking solutions and to approximate exposure times. Development andwashing of the strips is essentially the same as for aWestern blot analysis(Pierce Biotechnology, TechTip #24; Thermofisher). Such optimizationwill help prevent production of poor quality research data (see below)andalsohelpprevent problems such asweak signal, brief signal intensity,white bands on a high background or so called “reversed image”, and thepresence of a spotted or speckled background.

Although it is likely the most popular method, antisera probing ofthe Western blot in a sequential manner with washes in between isnot the only choice. Sundaram (1996) reported some time ago that itis feasible to add the primary and secondary antisera together in onestep saving time and the use of buffer solutions with no apparent lossof subsequent detection signal.

Wu, Stockley, and Martin (2002) has also analyzed Western blotdevelopment steps and found that inefficient washing is the primaryreason for high background in Western blots. The authors provideseveral troubleshooting tips such as lowering the non-ionic detergent(Tween-20) concentration in the antibody dilution buffer (0.02%–0.005%) and raising the concentration of the detergent in the washbuffer (0.5%). The authors also demonstrate that rapid blot washeswithdistilledwater or deionizedwater can be effective, likely due to its lowerionic strength promoting the removal of non-specific binding, withoutdamaging the membrane. A single wash in TBST (but with Tween-20 at0.5%) following thewater washes then restores the pH and proper ionicstrength prior to antisera incubation. My laboratory has also found thatthe temperature used during antibody incubation can have animportant influence on subsequent blot development. Some antiseraappear to work better (i.e. less background and more specificity) whenincubated on blots at 4 °C for up to 12 hwhile other antisera workmoreeffectively in room temperature conditions for short incubation periodssuch as1 h.Obviously, individuals should optimizewhatmay bebest forthe antisera they possess.

4.1. Polyclonal vs monoclonal

Bothmonoclonal and polyclonal antisera can be utilized forWesternblot analyses, but polyclonal antisera are more popular. There are

advantages and disadvantages in using either type. A prime advantage ofpolyclonal antisera can be that the sera can possess many antibodymolecules than can bind the target protein antigen of interest. The resultis that generally a stronger signal can ultimately be produced in theanalyses following secondary antisera incubation andblot development.In contrast, a major disadvantage can be that the sera can containantibodymolecules that canbind specifically to proteinsunrelated to theprotein of interest. Affinity purification can thus be an important step inantisera production for Western blots. Monoclonal antisera, by theirdesign, are much more sensitive and specific for the protein antigen ofinterest and this represents the biggest advantage of their utilization.One particular disadvantage can arise, however, if the specific antigenicsite is significantly affected (i.e. denatured) by electrophoresis condi-tions, thus limiting or preventing antisera interaction.

In total both antisera can be used for Western blot analyses but notall will work effectively. It is recommended that the investigator checkthe manufacturer's recommendations prior to investing considerablemoney on an antiserum and run a positive and negative control tissuelysate, in addition to the samples of interest, to verify the antiseracorrectly recognizes a protein of the predicted or known molecularmass. This point, in particular, is becoming more important as manycompanies are distributing a large number of antiseramade by a varietyof other companies and a purchasermay not be fully aware ofwhat theyare buying.My labhas found thatmanyantisera are not suitable, despiteclaims to the contrary, perhaps due to their inability to effectivelyrecognize epitopes following electrophoresis with denaturing reagents.

5. Detection and relative quantitation

Enhanced chemiluminescence or ECL has become the most popularmethod for detection inWestern blot analysis, although newdetectionshave emerged and others are being developed (Kurien & Scofield,2009a,b). The commercial development of chemiluminescent reactionsof specific reagents in Western blotting procedures was reviewedalmost 20 years ago (Durrant, 1990). It is a very sensitive method(Durrant, 1990; Dalessio and Ashley, 1992) and can be used toquantitate relative abundance of a protein(s) of interest unlikeenzyme-labeled antibodies utilizing soluble substrates that are con-verted into insoluble coloured endproducts (Kurien & Scofield, 2006).The principle is thatwhen a substrate such as 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) is oxidized by hydrogen peroxide, light isreleased during the chemical reaction (Fig. 2; Thorpe & Kricka, 1986;Thorpe, Kricka, Moseley, & Whitehead, 1985). The substrate is thus alimiting reagent and light production ends as the substrate is oxidized.The use of para-substituted phenolic compounds (e.g. 4-iodophenol) asenhancers in the reaction, where they replace luminol as the substrateand increase light output, has been incorporated into the procedure(Durrant, 1990).When aWestern procedure is properly optimized (e.g.antigen and antibody concentrations, see above) the linear responserange of the ECL reaction, over a certain window of time, will aid thequantitation of relative protein levels. It is also worth noting that manymanufacturers provide a variety of ECL-based Western blot detectionkits with specific detection abilities and users should consider what isbest for their individual needs and thoroughly optimize their protocolswith their choices. For example, detection kits with signal duration ofseveral hours (e.g. Pierce Supersignal West Pico, ThermoFisher; Amer-shamECL, GEHealthcare) that can be usedwithfilm areproduced aswellas extended duration substrate detection kits (e.g. Pierce SupersignalWest Dura, ThermoFisher; Amersham ECL Plus, GE Healthcare; Immun-Star, Bio-Rad) that produce a more intense signal for a longer duration(e.g. up to 24 h). The latter are generally designed for use with cooledcharged-coupled device cameras that are more sensitive, have a greaterresolution and a larger dynamic range of exposures than film.

With exposures captured on film or electronically under optimizedconditions, one can utilize densitometric methods to measure therelative quantities of a specific protein(s) on the blot compared to a

Fig. 2. A schematic of the ECL reaction and detection. Proteins adsorbed to membranesupport are recognized by the primary antisera followed by binding of HRP-conjugatedsecondary antisera to the complex. The horseradish peroxidase catalyzes a reactionleading to oxidation of phenolic enhancers that react with 5-amino-2,3-dihydro-1, 4-phthalazinedione (luminol). The end result is the production of light that can becaptured on film or by a digital imaging system.

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control lane (e.g. tissue positive control) or specific timepoint(e.g. 0 h) in an experiment. Film designed for ECL is still popular forsignal detection, although waning, and it is important that severalexposures are produced to ensure that the response is within thelinear range of the film. Keeping in mind the optimization stepsmentioned above, a frequent estimate used in our laboratory toproduce a good exposure is to try and produce an exposure with thesharpest appearing bands possible that still have a grayish appearanceand avoid exposures with bands that have indistinct or fuzzy edgesand are dark black indicating saturation of the film with light. Somecommercial software exists for image analysis of bands on film,whether protein or nucleic acid, but free software programs such asImage J , NIH Image (both available as freeware at (http://image.nih.gov/software/ip_packages.html)), and Scion Image (freeware atwww. Scioncorp.com) are popular choices. Digital imaging systemscontaining CCD cameras usually come packaged with proprietarysoftware designed for such analysis.

Once the appropriate exposures have been captured, blots areroutinely washed in a buffer such as TBST to remove any remainingdetection reagent. Following this or after gentle stripping of the blot(see below), it is worth considering archiving the wet blots in a Ziplocfreezer bag at −20 °C or −70 °C for future use. We perform thisregularly and have stored blots for years in old film boxes. This allowsa student or researcher to re-probe the blot several more times at amuch later date, for example as new reagents become available, andcan save time and precious samples.

6. Blot stripping

The idea of erasing a Western blot for subsequent re-probing wassuggested over two decades ago (Kaufmann, Ewing, & Shaper, 1987).At that time a 30-min incubation in 2% SDS (w/v) containing 100 mMβ-mercaptoethanol at 70 °C was reported as being able to removebound antisera and aid reuse of the blot. Presently, there are manycommercial sources of stripping solutions all promising the ability togently remove bound antisera and maintain protein adherence and

integrity for subsequent re-probing (e.g. ReView, Amresco; RestoreWestern Blot Stripping Buffer, Pierce Biotechnology; Western Re-probe, G-Biosciences; ReBlot, Millipore). The important step that mustbe undertaken with any of the above reagents, to ensure proper“erasure”, is to re-incubate the stripped blot in ECL substrate andexpose to film to ensure no protein detection occurs as a result ofremaining antisera. The blots can then be washed and archived or re-blocked for additional blot analyses.

As an alternative to stripping of blots, my lab will occasionallyanalyze one protein of interest (e.g. ∼30 kDa) and following detection,subsequently re-block the membrane and repeat the Western blotprobing and detection with a different antiserum that recognizes adifferent protein of considerably differentmolecularmass (e.g. 75 kDa).The caveat is that the user must be confident of the specificity andfidelity of the antiserum utilized, that is, each antibody specificallyrecognizes only the protein of interest on the blot.

7. Developments in fluorescent detection

Fluorescent probes for use inWestern blot analysis was purportedlyfirst reported by Fradelizi et al. (1998) and their use in Western blotprotein labeling is beginning tobecomemorepopular, althoughperhapsthe cost of the necessary equipment to image the signal may still besomewhat inhibitory to some laboratories. TraditionalHRP- andalkalinephosphatase (AP)-conjugated secondary antisera can be used forfluorescent immunodetection of proteins on Western blots in so calledenhanced chemifluorescence methods (ECF). In such situations,substrate reagents can be used to detect HRP-conjugates (AmplexGold, Emission: ∼535 nm, yellow fluorescence) or AP-conjugates(DDAO phosphate, Emission: ∼645 nm, far red fluorescence; Martin &Patton, 2003). However, the signals generated need to be visualizedwith UV epi-illumination and digital image capture. In these systems,the enzyme conjugated antibodies react with the substrates that, oncecleaved by the enzymes, produce a highly fluorescent endproduct. InECF methods, the signal is more stable and has a greater linear rangethan traditional ECL as well as compared to more direct fluorescencedetection methods (Amersham BioSciences Application Note, Code#63-0043-05, 2001). However, direct fluorescent detection of proteinsonWestern blots is simple, rapid, approaches the sensitivity of ECL, andstill possesses a greater quantifiable linear range than ECL detection(Gingrich, Davis, & Nguyen, 2000). The potentially great advantage ofdirect fluorescent detection lies in the ability to detect many differentfluorescent signals, termed multiplex analysis (Gingrich et al., 2000). Ituses differentfluorescent labels such as FITC, Cy3, and Cy5 conjugated tosecondary antisera in a manner similar to standard procedures inconfocal microscopy to analyze the sub-cellular localization of proteins.Multiplex analysis avoids the need to re-probe the blot. Recently,Delaive, Arnould, Raes, and Renard (2008) have reported that the use ofa three step protocol for fluorescence-basedWestern blot detection canmarkedly increase sensitivity of detection even further. This procedureutilizes a primary antisera step, a biotinylated secondary antisera step,and a tertiary fluorescently conjugated (e.g. CyDye or FITC) anti-biotinantibody step. The authors reported a 30 fold stronger signal fordetection thanwith afluorescently conjugated secondary antisera alone(two-step protocol). Importantly, both fluorescent and chemifluores-cent detectionmethods are easier to quantitate and visualize on CCD orlaser scanning imaging systems (Top et al., 2001).

Another type of probe that may prove popular in the future forfluorescent immunodetection of proteins on Western blots is quantumdots. These are semiconductor nanocrystals that can be excited by bluelight (488 nm) and fluoresce in narrow emission bands over a longrangeofwavelengthsdependingon the composition and size of the dots(e.g. Cadmium selenium, CdSe; Hibbs, 2004). The particles exhibit amarkedly decreased photobleaching capability and phototoxicity, butthe usefulness of these particles is most likely resident in the ability oftheir emission spectra to be tuned with one laser line. Thus,

Fig. 3. A list of some considerations for improving Western blot analysis.

176 D.J. MacPhee / Journal of Pharmacological and Toxicological Methods 61 (2010) 171–177

simultaneous or sequential detection of multiply-labeled antigens canbe conducted without any need for blot stripping. Makrides, Gasbarro,and Bello (2005) have recently demonstrated that quantumdots can beconjugated to antibodies opening the door for use of these particles inWestern blotting.

8. Summary

Since the development of the Western blotting method ∼30 yearsago, many protocol modifications and technologies have emerged forincreasing the sensitivity and speed of the method. Newer detectiontechnologies and more rapid analyses will continue to be offered butthere are still consistently similar and prime considerations that need tobe taken into account when conducting Western blot analysis (Fig. 3).For example, even emerging technologies will still require researchersto optimize lysis conditions, amount of protein loaded, antiseraconcentrations, membrane type and porosity, membrane backgroundfluorescence, washes, incubation times and exposure times. The endresult will hopefully be the production of the most accurate experi-mental data possible.

Conflict of interest statement

The author declares that he has no financial interest or conflict ofinterest in suggesting reagents for utilization from manufacturers ordistributors as the suggestions serve as a guide for investigators toexplore their own choices.

Acknowledgements

The authorwould like to thankDr. JulesDore (Division of BioMedicalSciences, Memorial University) for critically reading and commentingon the manuscript. Research programs by the author are supported by

theNatural Sciences andEngineeringResearchCouncil of Canada (Grant# #250218-07) and the Canadian Institutes of Health Research througha partnership with the Industrial Research and Innovation Fund(Government of Newfoundland and Labrador; Grant #ROP-82354).

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