VALIDATION OF A DOT-BLOT QUANTITATIVE TECHNIQUE

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JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 3, 91-97www.jpp.krakow.plN. GUILLEMIN1, B. MEUNIER1, C. JURIE1, I. CASSAR-MALEK1, J-F. HOCQUETTE1, H. LEVEZIEL2, B. PICARD1VALIDATION OF A DOT-BLOT QUANTITATIVE TECHNIQUE FOR LARGESCALE ANALYSIS OF BEEF TENDERNESS BIOMARKERSInstitute for Agricultural Research (INRA), UR1213, Herbivore Research Unit, Muscle Growth and Metabolism Group, ClermontFerrand/Theix Research Center, F-63122 Saint Genes Champanelle, France; 2Institute for Agricultural Research (INRA), UMR1061,Animal Molecular Genetics Unit, University of Limoges, IFR145, Sciences and Technique Faculty, F-87060 Limoges, France.1Beef tenderness is a very complex and multifactorial sensorial meat quality trait, which depends partly on musclecharacteristics. This tissue is very variable according to animal type (age, breed and sex) and rearing conditions.Consequently, beef tenderness exhibits a great variability. Different research programs have revealed several genes orproteins which could be good markers of beef tenderness. In order to validate the relation of these markers with beeftenderness on a large population of bovines, it is necessary to have a large-scale and trusty technique which can accessdifferent quantities of proteins related to tenderness. In this study we firstly compared Western-Blot and Dot-Blot.Secondly, we evaluated Dot-Blot technical and biological capabilities for the quantification of protein biomarkers. Theresults demonstrated that the Dot-Blot technique with fluorescence detection presents numerous interests. This techniqueallows a good reproducibility and permits the simultaneous analysis of a large number of samples. The Dot-Blot techniquedefined and validated in this study can be used for protein biomarkers analyses, notably to predict beef tenderness.Another major result of this study is that about 5 to 10 animals per group are required to detect large differences ( 1.5)in biomarker expression between tender and tough beef, whereas much larger numbers of animals (10 to 30) are requiredto detect smaller differences (about 1.2 to 1.3) taking into account the biological variability of these markers.K e y w o r d s : Dot-Blot, validation, large-scale analysis, biomarkers, protein quantification, immunologyINTRODUCTIONBeef is important in the human diet and represents animportant economical sector in different countries includingFrance (1). Consumers attempt high sensorial meat quality,especially tenderness (2). This meat quality trait depends onmuscle which is a very complex tissue. Indeed, musclescharacteristics like collagen and lipid contents, fibre typescontribute to a great extent to the variation in beef tenderness (3).In order to access to the variation in tenderness, severalprograms (4), based on biochemistry, proteomics,transcriptomics and genetics, have generated a list of at leastthirty potential beef tenderness biomarkers, at protein (5), RNA(6) and DNA levels (4). These potential biomarkers have beenrevealed by comparisons of animals groups which differ in thequality of the meat they produce.Larger analyses, on different muscles, animal types andbreeds, are necessary to confirm the roles of these thirtybiomarkers in the determinism of tenderness. This validationstep presented in this article concerns the protein level, in orderto develop in the future a protein prediction test for beeftenderness. For this, it is crucial to dispose of a reliable andlarge-scale analysis technique for protein quantification.To perform that kind of large-scale protein validation, somequantitative immunological techniques are available such asEnzyme-Linked Immunosorbent Assay (ELISA), Protein-array, orDot-Blot immunoassay. The ELISA technique is a fast quantitativemethod based on a solid support. For example it has beenemployed in the determination of the type I Myosin Heavy Chainin bovine muscle (7). However the weak point of ELISA is thattechnical conditions must be defined for each specific antibodyused. In the case of around thirty biomarkers to validate, it wouldrequire too much time to establish specific conditions for eachmarker. This is the reason why we did not selected ELISA for thevalidation of tenderness potential markers. Protein microarrayswere developed to study protein-protein interaction (8). Recently,some improvements of the technique allowed proteinquantification (9) and opened new ways to determine proteinquantities in a large scale analysis, notably for cancer orbacteriology researches. So, Protein-arrays represent a goodalternative for this study. However, the preliminary step beforeprotein quantification is to validate the specificity of antibodiesused on bovine muscles. This is routinely realised by Western-Blot.So, the chosen technique for protein quantification must be moresimilar as possible to Western-Blot, to be sure that antibody willreact with the same specificity in the high throughput technique asin Western-Blot. This is not the case for Protein-array, which havetechnical conditions (the support, incubation time and antibodyutilisation) quite different from those of Western-Blot. This is thereason why Protein-arrays were not selected for this study.Dot-Blot immunoassay was developed in 1982 (10) based onRNA Dot-Blot, and validated for large-scale absence/presence

92screening of protein markers in bacteriology, immunology andepidemiology researches. ELISA has been developed andtechnically validated 10 years before Dot-Blot for proteinquantification, and is nowadays a currently used technique.Because of technical specifications like proteins solubility, somestudies used Dot-Blot instead of ELISA (11). As exposedpreviously, the validation of an important number of differentproteins require usage of Dot-Blot instead of ELISA for alsotechnical and time-consuming reasons. Moreover, the Dot-Blottechnique is very similar to Western-Blot, and is a compromisebetween the advantages of Protein-array approach and those ofWestern-Blot technique. As antibody conditions (dilutions,incubation-time) are similar in Dot-Blot and Western-Blot, theantibody specificity can be guaranteed in a Dot-Blotquantification after a Western-Blot validation of the antibody,like the methodology employed by Duffy et al. (12). For allthese reasons, the Dot-Blot technique was chosen for this study.Because of the poor rate of Dot-Blot usage for proteinquantification, there is no technical and biological validation ofDot-Blot except for a precise use in a study context for one or twoproteins analysed (12, 13). So the validation of several differentprotein markers requires a more global Dot-Blot validation.This article presents the technical and biological validation ofDot-Blot as a large-scale technique for protein quantificationfrom bovine muscle total protein extraction. To achieve this goal,we have determined the inter- and intra- assay variability of DotBlot and the inter-assay variability of Western-Blot (Study 1), andthe Dot-Blot response for proteins of different characteristics(Study 2). We also present the biological validation of the Dotblot technique by demonstrating its capability to detect a wellknown muscle effect according the literature (Study 3). Finally,we illustrate a Dot-Blot application for the large scale validationof beef tenderness biomarkers (Study 4).MATERIALS AND METHODSChemical agents and apparatusAcrylamide, bisacrylamide, TEMED, ammonium persulfatewere from Amersham (Uppsala, Sweden). Chemical reagents,including urea, thiourea, DTT and CHAPS were from Sigma (St.Louis, MO, USA). The Mini-Protean electrophoresis apparatusand the Trans-Blot Cell were from Biorad (Hercules, CA, USA).The Minifold I Dot-Blot was from Schleicher&SchuellBioscience (Germany). The Odyssey Scanner was from LI-CORBiosciences (Lincoln, Nebraska, USA). The technical assayswere conducted with three antibodies corresponding to threepotential markers of beef tenderness according to previousresults: Heat Shock Protein 27 (14), Phosphoglucomutase andMyosin Binding Protein-H (15). Antibodies: anti-PGM and antiMyBP-H were from Santa Cruz (Santa Cruz, CA, USA), antiHsp27 was from AbNOVA (Taipei, Taiwan). Antibodiesspecificities were guaranteed on human proteins bymanufacturers. Secondary fluorescent antibody was from LICOR Biosciences (Lincoln, Nebraska, USA).Animals and samplesThis study was conducted with 18 Charolais young bullsfrom the INRA experimental program MUGENE (funded byANR and APIS-GENE through the GENANIMAL call). All theanimals were slaughtered at 15 or 19 months of age at theslaughterhouse of INRA in compliance with the current ethicalguidelines for animal welfare. Muscle samples from longissimusthoracis (LT) and semitendinosus (ST) were excised from eachanimal within 15 minutes after slaughter. Muscle samples wereimmediately frozen in liquid nitrogen and stored at -80 C untilprotein extraction. The tenderness value for each sample wasaccessed by mechanical (Warner-Bratzler test) analysis oncooked meat as described in Lepetit and Culioli (16).Total protein extractions were performed according toBouley et al. (17) from 13 LT and 8 ST frozen muscles fromyoung bulls in the denaturation extraction buffer (8.3M urea, 2Mthiourea, 1% DTT, 2% CHAPS). The protein concentration wasdetermined by spectrophotometry with Bradford assay (18).Protein extractions were stored at -20 C. A standard sample wasconstituted by mixing all samples from young bulls.Western-Blot membraneWestern-blot analysis were performed according to themethod previously described (19, 20) and slightly modified.Briefly, fifteen micrograms of total protein extraction samplewere separated in 1-D in a 12% polyacrylamide gel at 120 V, at4 C for 90 minutes in Mini-Protean apparatus. Proteins weretransferred to a PVDF membrane with Trans-Blot Cell apparatusat 210 mA at 4 C for 60 minutes. Membrane was blocked in a10% milk blocking buffer at 37 C for 20 minutes and thenincubated with the primary antibody. Infrared fluorescencedetection was then used for protein quantification.Dot-Blot membraneFifteen micrograms of total protein extraction sample weredeposited on a nitrocellulose membrane with Minifold I DotBlot apparatus. All samples were deposited by random order onthe 96-spots membrane. In addition, three protein quantities ofthe mixed standard sample (7.5 µg, 15 µg, 22.5 µg) weredeposited for data normalization. The Dot-Blot membrane wasair-dried for 5 minutes, and blocked in a 10% milk blockingbuffer at 37 C for 20 minutes and then incubated with theprimary antibody. Infrared fluorescence detection was then usedfor protein quantification.Antibodies incubationsThe specificity for each antibody used on bovine muscle waschecked by a Western-Blot with 15 µg of total protein extractionfrom bovine LT muscle per well, with a fluorescence detection at800 nm. The specificity was accessed by the detection of a singleband corresponding to the theoretical molecular weight of theprotein studied, like the method used by Duffy et al. (12). Themolecular weight was determined by the Western-Blot with BioradKaleidoscope ladder which emitted fluorescence at 700 nm. TheFig. 1 showed an example of this specificity validation on bovinemuscle for antibody anti-Hsp27 at 800 nm. After this step ofvalidation, antibodies can be used on bovine muscle as follow.The Western-Blot and Dot-Blot membranes were incubatedat 37 C during 90 minutes with the following concentrations(initial concentration/appropriate dilution): MyBP-H (1000µg/mL/1:4000), Hsp27 (200 µg/mL/1:3000), and PGM (1000µg/mL/1: 8000). Then the membranes were incubated at 37 Cfor 30 minutes with the anti-mouse fluorochrome-conjugatedLICOR-antibody IRDye 800CW (1 mg/mL). The first andsecond antibodies were diluted in 1% milk blocking buffer.Image analyses and protein quantificationAll Western-Blot and Dot-Blot were scanned with the OdysseyNIR imager, using the 800 nm laser, a 169 µm spatial resolutionand a fixed gain of 5. This infrared fluorescence detection wascharacterised as a quantitative and sensitive technique for proteinquantif t, contrary to Western-Blot. So, a possiblelimitation of Dot-Blot could be the accessibility of the protein bythe primary antibody. Low steric size proteins could be masked70Fig. 4. Variation in sample size required to detectsignificant differences in Hsp27 and MyBP-H amountsdepending of the expected differences to be detected.The number of animals per group to detect a significantdifference between two animal groups was calculatedaccording to the desired effect to be detected for twobiomarkers (Hsp27, MyBP-H) in bovine muscle taken intoaccount the biological variability which differs for the twomarkers. A pilot study (Table 2) was conducted on the twotenderness biomarkers (Hsp27 and MyBP-H) to determineintra-group standard deviation (i.e. the biologicalvariability which was 19 and 40% for Hsp27 and MyBPH respectively). The expected differences between groupmeans (i.e. the desired effect to be detected expressed as %of mean) and the biological variability entered statisticalpower calculation to estimate required sample size. Thusabout 30 samples per group are necessary to detect asignificant difference of 30% in MyBP-H protein content,but only less than 10 animals are necessary to detect asignificant difference of 30% in Hsp27 due to the lowerbiological variability of this second marker.by high steric size proteins on the spot surface, and so theprimary antibody can not find its epitope. The same problemcould be observed between low and high represented proteins,especially for this muscular samples, where 20% of the totalprotein mass is represented by actin only (29).The Dot-Blot experiments on three proteins (Hsp27, MyBPH and PGM) which differ according to their molecular weight,composition, three-dimensional structure and cellularabundance, showed in the study 2, that there were no technicallimitations for Dot-Blot with a large spectrum of differentproteins, from small size heat shock proteins to bigger enzymes.Moreover, the spot intensity was linearly correlated with thedifferent total protein quantities used (up to 22.5 µg), withoutsaturation or latency. So, Dot-Blot was proportional for thesedifferent quantities and can be employed for quantification with15 µg of total protein samples. This quantity is usually used inWestern-Blot to have a good representation of muscle proteins.To reproduce the Western-Blot technical conditions, 15 µg oftotal proteins were also used for Dot-Blot quantification. Theresults of Dot-Blot experiment in the study 3 with MyBP-Hrevealed a significant difference between ST and LT muscle,where MyBP-H quantity was higher in ST than in LT. Theseresults were in accordance with data cited from Gilbert et al.(25). So we can conclude that Dot-Blot was able to detect aknown protein quantity difference between two muscles.In the case of beef tenderness marker, the desired effects wewant to observe between two tenderness groups are generallyabout 20 or 30% of magnitude. To be statistically significant, the

96marker effect must be analysed on a sufficient number ofsamples per group. This number can be calculated by a poweranalysis using the T-test formula. This power analysis showedthat the number of required samples per group depends on thebiological variation of the studied marker and on the magnitudeof the effect to be detected. Therefore, for a desired effect of20% with a marker intra-group variability of 20% observed inthe study 4, like Hsp27, the required sample size group is 20.The same desired effect with a marker intra-group variability of40%, like MyBP-H would require a sample size group of 60. So,assessment of these markers requires large-scale analysis, whichis a strong advantage of Dot-Blot. This technique allows indeedthe analysis of the number of samples recommended by thepower analysis to have statistically significant results, rapidlyand easily.The large-scale abilities of Dot-Blot allow a higher numberof samples to be analysed in one experiment compared to theWestern-Blot technique. These have some consequences interms of results, because a large range of data is generated withinthe same experimental conditions (antibodies quantities,hybridization and revelation time). These have also aneconomical impact, because an experiment of Dot-Blot usesfewer amounts of antibodies and buffers, and is faster and easiercompared to Western-Blot. The large-scale abilities of Dot-Blotare however limited by the number of samples deposited onmembrane. Automatic membrane made by robot could enhancethe large-scale capacities of Dot-Blot. Because proteins are notpreviously separated according to their molecular weight andisoelectric point, it is not possible with Dot-Blot to distinguishand quantify specific isoforms of proteins like in proteomics.The Dot-Blot technique is a global protein approach lesssensitive than bi-dimensional electrophoresis or RT-PCR.Results from Dot-Blot are thus complementary with those ofproteomics, transcriptomics and genetics approaches.In conclusion, this work demonstrates that Dot-Blot istechnically as reliable as a Western-Blot, which is a standardtechnique employed for validation of protein biomarkers, for alarge spectrum of different proteins. In case of large-scaleanalysis requirement, Dot-Blot represents a good andcomplementary tool for protein biomarker validation. So, DotBlot is well-adapted for our tenderness biomarker validationstudy. In any case, the number of samples to be analysed must bepreviously calculated according to the biological variability ofthe studied biomarkers with potentially large differencesbetween markers.Acknowledgements: The PhD grant of N. Gu

Dot-Blot membrane Fifteen micrograms of total protein extraction sample were deposited on a nitrocellulose membrane with Minifold I Dot-Blot apparatus. All samples were deposited by random order on the 96-spots membrane. In addition, three protein quantities of the mixed standard sample (7.5 µg, 15 µg, 22.5 µg) were deposited for data .

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