Analytical Biochemistry 430 (2012) 108–110

Contents lists available at SciVerse ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier .com/locate /yabio

Notes & Tips

Comparison of Stain-Free gels with traditional immunoblot loadingcontrol methodology

Alex D. Colella a,⇑, Nusha Chegenii b, Melinda N. Tea a, Ian L. Gibbins c, Keryn A. Williams a, Tim K. Chataway b

a Department of Ophthalmology, Flinders University, Bedford Park, SA 5042, Australiab Flinders Proteomics Facility, Department of Human Physiology, Flinders University, Bedford Park, SA 5042, Australiac Department of Anatomy and Histology, Flinders University, Bedford Park, SA 5042, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 July 2012Received in revised form 16 August 2012Accepted 16 August 2012Available online 26 August 2012

Keywords:ImmunoblotLoading controlStain-Free gelWestern blotSemiquantitative

0003-2697/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ab.2012.08.015

⇑ Corresponding author.E-mail address: alex.colella@flinders.edu.au (A.D. C

1 Abbreviations used: LC, loading control; SF, Staindifluoride; TBS, Tris-buffered saline; PHD-2, Prolyl hydperoxidase; OD, optical density; pre-chemi 1/2, pre-with first/second antibody; SNR, signal-to-noise ratio;tion; ANOVA, analysis of variance.

Loading controls are necessary for semiquantitative Western blotting to compensate for loading errors.Loading control methods include the reprobing of membranes with an antibody against a constitutivelyexpressed protein or staining the membrane with a total protein stain. We compared the loading controlperformance of recently released Stain-Free (SF) gels with Sypro Ruby (SR) and reprobing using b-actin.SF gels demonstrated superior performance in that they were faster, required fewer steps and consum-ables, and allowed the quality of electrophoresis and Western transfer to be assessed before committingto costly and time-consuming Western blots.

� 2012 Elsevier Inc. All rights reserved.

Quantitative Western blotting is a widely used method for mea-suring the relative abundance of target proteins. To allow for arti-facts resulting from pipetting inaccuracy, imprecise proteinestimation, or uneven transfer of proteins, a loading control (LC)1

is usually employed. The most frequently used LC uses an antibodythat detects a constitutively expressed protein such as b-actin. Themeasurement of the total protein transferred to membranes is alsoused as an LC, using total protein stains such as Ponceau S [1], AmidoBlack [1], Sypro Ruby [1–3], and Coomassie Blue [4]. However, bothtotal protein stains and constitutive protein Western blot LC meth-ods have drawbacks. The use of a single protein LC typically adds sig-nificant time and cost to experiments. Furthermore, it must beestablished that the LC protein does not change as a consequenceof the experimental variable under test and does not saturate thedetection system. There are also instances where a suitable LC pro-tein is not available or appropriate, for example, when comparingabundance of a specific protein in different tissue extracts wherethe LC protein concentrations may differ. Although membraneprotein stains such as Sypro Ruby do not suffer from the issues

ll rights reserved.

olella).-Free; PVDF, polyvinylideneroxylase 2; HRP, horseradish

chemiluminescence detectionR2, coefficient of determina-

associated with the use of single protein LCs, they do add significantcost and time to semiquantitative Western blotting experiments.

Stain-Free (SF) gels contain proprietary trihalo compounds thatreact with proteins and are detectable by CCD (charge-coupleddevice) imagers. Trihalo compounds have been shown to reactwith tryptophan residues using an ultraviolet light-induced reac-tion to produce fluorescent light [5]. SF imaging adds approxi-mately 5 min to the Western blotting procedure and provides analternative to both LC proteins and Sypro Ruby. In this study, wecompared the performance of SF gels with Sypro Ruby and theuse of LC proteins to determine whether the novel technologycould provide similar performance as an LC.

Retinas from 6-day-old rat pups were dissected as described byTea and coworkers [6], and the protein content was purified usingTRIzol reagent following the manufacturer’s protocol. The purifiedprotein pellet was dissolved in 4� Laemmli’s sample buffercontaining 4 M urea and 50 mM dithiothreitol. The concentrationof the protein preparation was determined using an EZQ assay(Invitrogen).

A series of replicate Western blots containing protein loadsranging from 10 to 40 lg were analyzed in triplicate on CriterionTGX Stain-Free Any kD 18 well comb, 30 ll, 1-mm precast gels(Bio-Rad), following the manufacturer’s protocol. Proteins werethen transferred to Immun-Blot LF polyvinylidene difluoride(PVDF) membrane, 0.45 lm (Bio-Rad), using a Turbo Blot transferunit (Bio-Rad).

Fig.1. Total protein membrane imaging and antibody detection. Increasing protein loads (10, 17.5, 25, 32.5, and 40 lg of protein) derived from rat retinas were separated onSF polyacrylamide gels, transferred to low-fluorescence PVDF membrane, and imaged sequentially. (A) SF total protein post-transfer with a representative transect. (B) SFtotal protein prior to detection of PHD-2 antibody. (C) SF total protein prior to detection of b-actin antibody. (D) Sypro Ruby total protein stain post-transfer. (E) ECL detectionof PHD-2. (F) ECL detection of b-actin. (G) Standardized OD measurements of SF membranes (n = 7) imaged post-transfer (solid circles), imaged prior to detection of PHD-2antibody (pre-chemi 1; solid triangles), imaged prior to detection of b-actin antibody (pre-chemi 2, crosses), and from Sypro Ruby total protein stained membranes (n = 3;squares) were standardized to allow for a comparison of the different protein images. Averaged OD values were standardized by dividing the mean of technical replicates bythe mean of all samples from the same blot, and then the means of all standardized values were plotted.

Notes & Tips / Anal. Biochem. 430 (2012) 108–110 109

Stain-free imaging was performed using a ChemiDoc MP imager(Bio-Rad) with a 1-min stain activation time and 3- to 7-s exposuretimes for all subsequent membrane protein images. Total proteinimages were obtained pre-blocking, post-blocking, and prior tothe chemiluminescent detection of antibodies. Where total proteinwas visualized using Sypro Ruby stain (Invitrogen), SF gels weretransferred to low-fluorescence PVDF membrane (Bio-Rad) withno activation of the SF dye by ultraviolet exposure. After stainingaccording to the manufacturer’s instructions, Sypro Ruby proteinblot stain (Invitrogen) stained membranes were imaged using aChemiDoc MP imager (Bio-Rad).

Membranes were blocked in 5% skim milk/Tris-buffered saline(TBS)/0.1% Tween 20 (1 h, room temperature). All primary antibod-ies were diluted in 2.5% skim milk/TBS/0.05% Tween 20. Mem-branes were first probed with anti-Prolyl hydroxylase 2 (PHD-2,D31E11 monoclonal antibody rabbit, cat. no. 4835, Cell Signaling),diluted 1:1500, and incubated overnight at 4 �C. Horseradish per-oxidase (HRP)-linked monoclonal anti-rabbit secondary antibodies(Jackson ImmunoResearch), diluted 1:2000, were incubated atroom temperature for 1 h. Chemiluminescence from the reactionof HRP-linked secondary antibodies and Immun-Star WesternCsolution (Bio-Rad) was captured digitally using a ChemiDoc MP im-ager (Bio-Rad). Membranes were either treated with H2O2 (n = 3)to inactivate the HRP-linked secondary antibodies bound to themembrane as described by Sennepin and coworkers [7] or werestripped (n = 7) using 0.1 M glycine, 20 mM magnesium acetate,and 50 mM KCl (pH 2.2) before being reblocked. Membranes werereprobed with anti-b-actin antibody (cat. no. A00730, GeneScript),diluted 1:2000, and incubated overnight at 4 �C. Membranes werethen incubated with HRP–donkey anti-mouse IgG (H+L) monoclo-nal secondary antibodies (cat. no. 715-035-150, Jackson Immuno-Research), diluted 1:3000, at room temperature for 1 h.

All images were analyzed using Image Lab 4.0.1 software (Bio-Rad). For relative quantification of total protein stains, transects

through the center of the lane running from top to bottom wereused (Fig. 1A) and global background subtraction of equal-sizedtransects, placed in a blank area of the membrane, was performed.The lane volumes (optical densities, ODs) for each protein load onthe SF gels were monitored at the following stages of the work-flow: 1, post-transfer; 2, post-blocking (Fig. 1A); 3, pre-chemilumi-nescence detection with first antibody (pre-chemi 1, Fig. 1B); 4,post-stripping or H2O2 treatment and reblocking; 5, pre-chemilu-minescence detection with second antibody (pre-chemi 2;Fig. 1C). For calculation of coefficients of determination, blots werestandardized so that data from SF and Sypro Ruby protein stains, aswell as antibody signals, could be compared. This was achieved bydividing mean values of each technical replicate by the mean of allsamples from the same blot. When combining data from multipleblots, the mean of standardized values for each replicate serieswas calculated. A linear increase in OD, associated with increasingprotein load, was maintained through the workflow, althoughsome deviation was seen for the SF gels at a 40-lg load (Fig. 1G).

To gain a statistical measure of linearity, coefficients of determi-nation (R2) were calculated for each of the SF images as well as forSypro Ruby and the two antibodies used (Fig. 2). Blots were stan-dardized so that data from SF and Sypro Ruby protein stains, aswell as antibody signals, could be compared. This was achievedby dividing mean values of each technical replicate by the meanof all samples from the same blot. When combining data from mul-tiple blots, the mean of standardized values for each replicate ser-ies was calculated. Values for each stage of gel processing werecompared with repeated measures analysis of variance (ANOVA)using SPSS version 19 (SPSS). Statistical analysis revealed no signif-icant difference between the measurements for any of the proteinloads: repeated measures ANOVA, F(7,42) = 1.4, P = 0.3.

High R2 values were obtained for all OD measurements taken,with the lowest value (0.86) being obtained for SF total proteinmeasured prior to the chemiluminescent detection of the second

Fig.2. Regression analysis of membrane total protein and antibody signals. Coef-ficients of determination (R2) analysis was calculated for SF, Sypro Ruby, PHD-2, andb-actin ODs. R2 values for b-actin were calculated separately for membranes thatwere stripped (n = 7) and H2O2 treated (n = 3). There were no significant differencesbetween R2 values for any of the total protein or antibody signals: repeatedmeasures ANOVA, F(7,35) = 1.5, P = 0.2.

110 Notes & Tips / Anal. Biochem. 430 (2012) 108–110

antibody (b-actin) and the highest value being obtained for SF totalprotein pre-blocking (0.98). No significant difference in linearitywas observed among SF, Sypro Ruby, PHD-2, and b-actin: repeatedmeasures ANOVA, F(7,35) = 1.5, P = 0.2. The R2 values for b-actinwere calculated separately for blots that were stripped (n = 7)and those that were H2O2 treated (n = 3) to ascertain how thesetwo treatments may have affected the linearity of the signal pro-duced. The H2O2 inactivation of membrane-bound HRP-linked sec-ondary antibodies was shown by Sennepin and coworkers [7] toenable the reprobing of single membranes at least five times with-out stripping. However, no quantitative analysis was performed inthis study. The advantage of the H2O2 method lies in its simplicityand speed. Furthermore, it does not use harsh stripping conditionsthat can lead to loss of protein bound to PVDF membranes [7]. Nosignificant difference in linearity was observed between blotsreprobed with b-actin after either stripping or H2O2 treatment(stripping R2 = 0.91, H2O2 R2 = 0.87, P > 0.05), indicating that H2O2

treatment provides similar performance to low pH membranestripping.

The signal-to-noise ratio (SNR) was calculated for each of theblotting steps: pre-blocking, post-blocking, pre-chemi 1, and pre-chemi 2 for the 25-lg load. Each handling step was found to de-crease the SNR. Blocking of membranes post-transfer reduced theSNR from 2.4 to 1.7. Incubation of membranes with primary andsecondary antibodies and their associated washes reduced theSNR to 1.2 with the SNR prior to the second enhanced chemilumi-nescence (ECL) exposure being 1.1 but did not significantly affectthe coefficient of determination values.

Our results indicate that SF gels are an alternative to existing LCmethods. We found that the quantification of protein load for SFgels was linear from 10 to 40 lg, making this methodology suitablefor compensation of loading errors on Western blots with proteinloads up to 40 lg. Both SF and Sypro Ruby provide equivalent per-formance as an LC, as shown by the similar R2 values calculated forall of the LCs tested.

We also examined the SNR of the labeled SF proteins at eachstage of the Western blot, including the reprobing of the membrane.Not surprisingly, the SNR of SF blots decreased with each manipu-lation of the membrane, although the linearity of the SF signals wasnot significantly decreased despite the increase in background sig-nal. In our experience, the best point at which to measure the OD ofSF blots is prior to blocking of membranes because the SNR is great-est at this point. Greater care must be taken when measuring ODs atlater stages in the Western workflow because strongly fluorescingartifacts are often introduced as a result of membrane handling.

There are several benefits to using the SF system in comparisonwith Sypro Ruby and antibody-based LC methods, with the princi-pal benefit being the reduction in the time required to perform aquantitative Western blot of 1.5 h compared with Sypro Rubyand by 1 day compared with antibody detection of a constitutivelyexpressed protein. There are also cost savings in reagents with SFgels compared with antibody LCs. In addition, SF gels can confirmthe quality of electrophoresis and Western transfer before commit-ting to the application of antibodies and detection reagents forWestern blots. The SF system requires a lower number of handlingsteps and, therefore, is subject to potentially fewer procedural er-rors when compared with Sypro Ruby and antibody LC methods.

In conclusion, we found that the SF technology produces anoverall superior performance as an LC compared with Sypro Rubyand antibody LCs because Western blot experiments can beaborted if a gel or membrane is found to have resolved or trans-ferred in a defective manner, saving considerable time and ex-pense; reduces the time needed because no membrane stainingis required and LC antibody blotting and prior optimization is notnecessary; and reduces costs because the need for expensive addi-tional reagents such as Sypro Ruby and other Western blotting re-agents is negated. Therefore, SF gels provide a faster and morecost-effective alternative to traditional methodologies.

Acknowledgments

Keryn A. Williams is supported by the National Health andMedical Research Council (NHMRC). This work was supported bythe Ophthalmic Research Institute of Australia and the FlindersMedical Centre Foundation.

References

[1] G.M. Aldridge, D.M. Podrebarac, W.T. Greenough, I.J. Weiler, The use of totalprotein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting, J. Neurosci. Methods 172(2008) 250–254.

[2] M. Hagiwara, K. Kobayashi, T. Tadokoro, Y. Yamamoto, Application of SYPRORuby- and Flamingo-stained polyacrylamide gels to Western blot analysis, Anal.Biochem. 397 (2010) 262–264.

[3] C.S. Stoner, G.D. Pearson, A. Koc, J.R. Merwin, N.I. Lopez, G.F. Merrill, Effect ofthioredoxin deletion and p53 cysteine replacement on human p53 activity inwild-type and thioredoxin reductase null yeast, Biochemistry 48 (2009) 9156–9169.

[4] C. Welinder, L. Ekblad, Coomassie staining as loading control in Western blotanalysis, J. Proteome Res. 10 (2011) 1416–1419.

[5] R.A. Edwards, G. Jickling, R.J. Turner, The light-induced reactions of tryptophanwith halocompounds, Photochem. Photobiol. 75 (2002) 362–368.

[6] M. Tea, R. Fogarty, H.M. Brereton, M.Z. Michael, M.B. Van der Hoek, A. Tsykin,D.J. Coster, K.A. Williams, Gene expression microarray analysis of early oxygen-induced retinopathy in the rat, J. Ocul. Biol. Dis. Inform. 2 (2009) 190–201.

[7] A.D. Sennepin, S. Charpentier, T. Normand, C. Sarre, A. Legrand, L.M. Mollet,Multiple reprobing of Western blots after inactivation of peroxidase activity byits substrate, hydrogen peroxide, Anal. Biochem. 393 (2009) 129–131.