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Development of pan-specific antibody against trimethyllysine for protein research Liang, Ziqian; Wong, Ronald P; Li, Lin H; Jiang, Hesheng; Xiao, Hao; Li, Gang Jan 22, 2008

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ralssBioMed CentProteome ScienceOpen AcceMethodologyDevelopment of pan-specific antibody against trimethyllysine for protein researchZiqian Liang†1, Ronald PC Wong†2, Lin Hong Li1, Hesheng Jiang1, Hao Xiao*1 and Gang Li*2Address: 1Rehabilitation Center of Burns and Plastic Surgery and Medical Research Center, Guangxi Medical University, Nanning, China and 2Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, CanadaEmail: Ziqian Liang -; Ronald PC Wong -; Lin Hong Li -; Hesheng Jiang -; Hao Xiao* -; Gang Li* -* Corresponding authors    †Equal contributorsAbstractBackground: Trimethylation of the Nε-lysine residues in a protein is one of the most importantevents of posttranslational modifications. Simple methods for rapid detection and isolation of theNε-trimethylated protein species are needed. This report introduces a novel method to preparethe affinity purified antibody specific for the Nε-trimethylated lysine (tMeK). The applications of thepurified antibody are also reported in this paper.Methods: We generated the methylated keyhole limpet heomocyanin (KLH) under controlledchemical methylation reaction using CH3I and used it as an immunogen to raise anti-methylatedlysine antibodies. The tMeK specific antibody was selectively isolated using a two-step affinitychromatography in which the mMeK/dMeK specific antibodies were removed and the tMeKspecific antibody was captured. Finally, the eluted anti-tMeK antibody was characterized.Results: The ELISA results indicated that the antibody reacted only to tMeK but not to mono- anddimethyllysine. Western-blot results showed that the Nε-trimethylated proteins were detected inboth animal tissue and cultured cells and that the antibody signal could be competitively inhibitedwith free tMeK.Conclusion: The specific tMeK antibody we developed is useful for one-step isolation of proteinswith Nε-trimethyllysine residues and also for the detection, identification and localization ofproteins with trimethyllysine residues in the cells.BackgroundSimilar to protein phosphorylation and acetylation, pro-tein methylation is one of the most important post-trans-lational modifications in regulating protein functions.tant role in the regulation of chromatin structure, geneexpression and DNA damage [1,2]. Histones and p53could be enzymatically methylated by a family of proteinlysine methyltransferases [3,4] and demethylated enzy-Published: 22 January 2008Proteome Science 2008, 6:2 doi:10.1186/1477-5956-6-2Received: 5 September 2007Accepted: 22 January 2008This article is available from:© 2008 Liang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 8(page number not for citation purposes)Perhaps, histone is the best known target for proteinmethylation. Histone lysine methylation plays an impor-matically by a family of demethylases [5,6]. The singlelysine residue could exist as mono-, di-, tri-methylatedProteome Science 2008, 6:2 unmethylated forms with different functional conse-quences [7]. Trimethylation of a specific lysine residue inhistone H3 is reported to be associated with inactive Xchromosome [5,8,9].Most of the reports on protein methylation are related tohistone methylation and a few of them related to the p53methylation [10]. Other methylated proteins are stilllargely unknown. As protein methylation may be associ-ated with embryonic development and diseases [1], theproteomic survey of the methylated protein patterns indifferent developmental stages and human diseasesbecome an important area in epigenetic research. In thispaper, we report a novel method to generate and purify apan-specific, Nε-trimethyllysine antibody (anti-tMeK),which could be used as a simple tool for the study of pro-tein trimethylation profiles. We generated the methylatedkeyhole limpet heomocyanin (KLH) under controlledchemical methylation reaction using CH3I and used it asan immunogen to raise anti-methylated lysine antibodies.The tMeK specific antibody was selectively isolated usinga two-step affinity chromatography in which the mMeK/dMeK specific antibodies were removed and the tMeK spe-cific antibody was captured. Finally, the eluted anti-tMeKantibody was characterized and we demonstrated that theanti-tMeK antibody could be used as a functional tool forthe detection of the trimethylated proteins using Westernblot and immunofluorescence and isolation of trimethyl-ated proteins using immunoprecipitation.ResultsPurification and ELISA characterizationIdeally, a trimethyllysine-specific antibody (anti-tMeK)should be highly specific and has strong affinity to tri-methyllysine (tMeK) but not to monomethyllysine(mMeK) and dimethyllysine (dMeK). The rationale ofdeveloping such an antibody is to use a synthetic trimeth-ylated protein, in which, the Nε-amino group of the lysineside chain was trimethylated, as an immunogen. Methyl-ation of the ε-amine groups of a protein using iodometh-ane (CH3I) generates mono-, di- and trimethylated lysineresidues in a protein (Figure 1a). The yield for trimethyl-ated species was increased by performing the reaction athigher pH and in prolonged reaction time (see Materialsand Methods). The highly immunogenic protein, KLH,was used for the methylation reaction. The dialyzed meth-ylated KLH was immunized to the rabbit to generate anti-methyllysine antibodies. After 60 days from initial immu-nization, immune serum was prepared and ELISA test wascarried out to test the titer. The serum has 50% maximumOD titer at 1:50,000 to tMeK-BSA conjugates, approxi-mately 1:2,000 and 1:10,000 to mMeK-BSA and dMeK-BSA conjugates respectively (data not shown). The meth-primary ELSIA results indicated that the anti-methylatedKLH immune serum cross-reacted with each form of themethylated lysine peptides.The rabbits immunized with methylated KLH containedspecific antibodies against all the methylated species. Thestrategy for isolating and purifying the tMeK-specific anti-body is illustrated in Figure 1b. The immune serum wasGeneration of anti-trimethyllysine antibodyFigure 1Generation of anti-trimethyllysine antibody. A. The reaction scheme for chemical synthesis of KLH with trimethyllysine residues. B. The scheme for strategic affinity purification of the trimethyllysine-specific antibody. The antibody in serum cross-reacted to monomethyllysine and dimethyllysine is removed by the mMeK/dMeK affinity column. The remaining trimethyllysine-specific antibody in the serum is further puri-fied using tMeK affinity column.Page 2 of 8(page number not for citation purposes)ylated KLH was synthesized while the mMeK, dMeK andtMeK used in ELISA were purchased commercially. Theinitially passed through a mMeK/dMeK affinity column.The antibodies bound to the column was eluted andProteome Science 2008, 6:2 to be about 10 mg. SDS-PAGE staining sug-gested the eluted proteins were mainly IgG with over 90%purity (data not shown). The yield for mMeK/dMeK anti-bodies was approximately 50 µg/mL of serum.After removal of the mMeK/dMeK antibodies, theimmune serum flowed through the mMeK/dMeK affinitycolumn was then passed through the tMeK affinity col-umn. The antibody bound to the tMeK column was elutedand determined to be approximately 50 mg. SDS-PAGEstaining indicated that the IgG purity is over 90% (datanot shown). The yield for tMeK-specific antibodies wasapproximately 250 µg/mL.The specificity of the purified anti-trimethyllysine toimmobilized mMeK-, dMeK- and tMeK-BSA was deter-mined by indirect ELISA (Figure 2). BSA is used here tofacilitate the immobilization of the small methyllysinemolecule onto the plastic surface of the micro plate. Thechemical reaction for the preparation of three forms ofmethyllysine-BSA conjugates is identical. The primaryantibody dilution buffer also contains 1% of BSA. TheELISA results showed that the tMeK-specific antibody didnot react well with both mMeK and dMeK-BSA butstrongly to tMeK.Immunoblotting detection and immunoprecipitation of the proteins with Nε-trimethyllysine residues in cell culture and animal tissueFigure 3a showed that the anti-tMeK antibody recognizedvarious proteins in the human melanoma cells. Thoseprotein bands recognized by the anti-tMeK antibodycould be completely inhibited by the free tMeK suggestingthat the antibody is Nε-trimethyllysine-specific and theprotein bands recognized by the anti-tMeK are trimethyl-ated.There are 7 major protein bands (14, 17, 19, 30, 32, 40,and 54 kD) recognized by anti-tMeK antibody accordingto the Western blot (Figure 3a). The strongest bands rec-ognized by tMeK-specific antibody were 17 kD and 40 kD.The film exposure time for the blot was less than 1 min.Some of the proteins with lower expression recognized bythe antibody may not be revealed under such conditions.Since lysine residues of histones can also be acetylated, wetested if the histone deacetylase inhibitor trichostatin A(TSA) would have an effect on trimethylation of cellularproteins. Our data showed that TSA increased the trimeth-ylation level, particularly for the 14 kD and 18 kD pro-teins.In the mouse spleen lysate, approximately 5 major proteinbands were recognized by the anti-tMeK antibody. Themethylated protein pattern is somewhat similar tomelanoma cell lysate. The major bands recognized are 14,17, 30, 54, and 70 kD (Figure 3b). The protein bands rec-ognized by the antibody could also be completely inhib-ited by free tMeK.Immunoprecipitation in undenatured condition is impor-tant for protein isolation and purification. Approximately200 µg of anti-tMeK antibody was utilized to isolate pos-sible trimethylated proteins. Using the anti-tMeK HRPconjugates for the Western blot analysis of the anti-tMeKimmunoprecipitated proteins, the massive interference ofIgG bands produced by the secondary anti-rabbit IgG HRPcould be eliminated. No IgG signal was observed in thecompetitive inhibition by tMeK BSA (Figure 3b). TheWestern blot results however, showed different pattern ofproteins. In the crude lysate, only 5 major protein bandswere recognized by the anti-tMeK HRP while the signal inthe crude lysate was significantly enriched in the IP sam-ple. However, the 70 kD proteins detected in crude lysatecould not be isolated by anti-tMeK antibody (Figure 3b).In the IP sample, anti-tMeK could capture more than 6protein bands with 14–16, 17, 20–23, 30, 40, and 54 kD.The protein band at 70 kD was only captured by tMeK incrude lysate. The 20–23 kD, 30 kD proteins were signifi-cantly enriched while the 20–23 kD bands were not visi-ble in the crude lysates. Apparently all the IP proteinbands were blocked with the presence of free tMeK mole-cule, suggesting that the IP proteins contained tMeK-resi-dues.The specificity of the tMeK-purified antibody in an ELSIA testFigure 2The specificity of the tMeK-purified antibody in an ELSIA test. The ELISA reaction of the purified tMeK-specific anti-body was carried out with immobilized tMek-BSA, mMeK-Page 3 of 8(page number not for citation purposes)To confirm whether the proteins immunoprecipitated bythe antibody we generated are indeed trimethylated, weBSA, or dMeK-BSA.Proteome Science 2008, 6:2 three well established site-specific anti-trimethylly-sine antibodies against histone H3 (H3K4me3,H3K9me3, H3K27me3) for Western blotting. Figure 3cshows that all three trimethylated species of histone H3could be detected in the proteins immunoprecipitatedwith our antibody.Immunofluorescent stainingUsing the human melanoma cells as working model, theFITC conjugated anti-tMeK antibody could strongly stainthe nuclei of the cell and weakly in the cytoplasm. Brighterspots of the staining could also be observed in the nuclei(Figure 4). The fluorescent staining signal could be signif-icantly inhibited with the presence of the tMeK, suggest-ing that staining signal is trimethyllysine-specific.Discussion and ConclusionProtein methylation is an important event of post-transla-tional modification. The goal of proteomic studies is toreveal that how the protein modification plays its role ingene expression, protein interaction and signal transduc-tion. To understand the roles of protein methylation, sim-ple and rapid methods for detection, identification,methylated proteins is one of the important aspects tofacilitate the proteomic survey of the protein methylation.The pan-specific antibodies against mMeK and dMeKhave been commercially available. The purpose of thisresearch is to develop a specific antibody against tMeKwhich could be applied to the proteomic survey and to thebasic research of bioscience.In our previous report [11], we used modified protein asimmunogen and antibody against single modified aminoacid could be raised and isolated through specific immu-noaffinity chromatography. To produce an antibodyagainst single molecule of trimethyllysine, a trimethylatedprotein may be the best choice as immunogen. Enzymaticsynthesis of trimethylated protein may not be a goodchoice for generation of such immunogen as the enzymereaction is highly site-specific. Chemically trimethylatedproteins may produce desired epitopes in an immuno-genic protein. Reductive methylation of proteins usingformaldehyde followed by mild reduction withcyanoborohydride makes this possible. However, theproducts mainly consist of mono- and di-methylatedlysine instead of trimethyllysine [12]. We therefore soughtWestern blot and immunoprecipitation analysis of proteins from human melanoma cells using the anti-tMeK antibodyFigure 3Western blot and immunoprecipitation analysis of proteins from human melanoma cells using the anti-tMeK antibody. A. Cells treated with or without TSA were lysed for Western blot (50 µg/lane) using the trimethyllysine-purified antibody, with and without the presence of free tMeK. B. Western blot analysis of the anti-tMeK immunoprecipitated proteins from the crude lysate of the mouse spleen using anti-tMeK HRP conjugates (0.25 µg/ml). The signal was competitively inhibited by the free tMeK (10 µg/ml). C. Western blot analysis of immunopreciptates by IgG control or anti-tMek antibody with H3K4me3, H3K9me3, or H3K27me3 antibodies.Page 4 of 8(page number not for citation purposes)isolation and localization of the methylated proteins isneeded. Development of an antibody specific for theto use CH3I to methylate the Nε-amino group of lysineresidues in a protein at high pH (pH > 9). Primary and sec-Proteome Science 2008, 6:2 amines are readily methylated under this condi-tion. The Nε-methylation products are controlled by thepH, time and temperature of the reactions. A conven-tional high immunogenic protein KLH was methylatedwith CH3I at pH > 9, 35°C for more than 6 h. Presumably,the higher in pH and temperature, longer reaction time,the more trimethylamine species will be generated. Theresulting methylated immunogen may yield more tri-methylated protein specific antibodies.The initial ELISA titration of the crude serum indicatedthat the population of the antibodies against KLH meth-ylated by CH3I has higher affinity for the trimethyllysine(tMeK). Subsequently, the immunoaffinity chromatogra-phy also demonstrated that our rationale is correct.Approximately 80% (50 mg) of the methyllysine specificantibodies specifically bind to tMeK, while only about20% (10 mg) of the antibodies bind to mMeK/dMeK col-umn. The ELISA data (Figure 2) showed that the antibodypurified with tMeK (anti-MeK) only recognized the tMeK-BSA, not mMeK- or dMeK-BSA, suggesting that the anti-The ELSIA data provide us the information that this anti-body is specific for tMeK not mMeK or dMeK. Usinghuman melanoma cell culture as working model, Westernblot analysis indicated that multiple proteins with differ-ent molecular weight range from 14 kD to 54 kD couldeasily be detected with the anti-tMeK-specific antibody(Figure 3a). Similar finding was also observed when theantibody was applied to the protein extract of the mousetissue extract (Figure 3b). The protein bands recognizedby this tMeK-specific antibody could be completely inhib-ited by the presence of free tMeK, suggesting the antibodycould be specifically used for detection of the proteinswith tMeK residues. The best known trimethylated pro-teins are histones, particularly the histone H3 [2,13,14].The Western blot results of both cultured cells and animaltissue indicated that the 17 kD band may correspond tohistone H3. In addition, using the well established site-specific anti-trimethyllysine antibodies against histoneH3 confirmed the presence of histone H3 in the immuno-precipitates isolated with our antibody. There are manyprotein bands recognized by anti-tMeK not only in West-ern blot but also in immunoprecipitation. The resultsimply that many other trimethylated proteins remain uni-dentified.In order to study the trimethylation profile of the pro-teins, methods for rapid isolation of the trimethylatedspecies is needed. Immunoaffinity chromatography or IPwith anti-tMeK is one of the most efficient methods forthis purpose. Immunoprecipitation studies (Figure 3b&3c) showed that this tMeK specific antibody could cap-ture the trimethylated species efficiently. Most of the tri-methylated proteins in the crude lysate recognized in theWestern blot detection system could also be specificallyisolated from the crude lysate by immunoprecipitationand some of them could be significantly enriched byimmunoprecipitation implying the potential use of thisanti-tMeK antibody in isolating trimethylated proteins forproteomics study.Immunofluorsecent staining of human melanoma cellswith this anti-tMeK showed strong staining in the nuclei(histone associated) and weak signal in the cytosol. Theimmunofluorescent signal of the antibody was blocked byfree tMeK, suggesting the antibody staining signal ishighly specific for tMeK. The immunofluorscent stainingresult correlates well with the fact that the lysine residuesof histones could be trimethylated at multiple sites andthe expression level of histones is usually high and stable.The bright spots of the nuclear immunofluorescent stain-ing could be explained by the fact that some histones asso-ciated with the chromosome maybe heavily trimethylated[8]. Those spots may also be the heterochromatin inImmunofluorescence staining of the human melanoma cells using tMeK-spe ific an ibodyFigure 4Immunofluorescence staining of the human melanoma cells using tMeK-specific antibody. Cells treated with or without TSA (200 ng/ml) for 16 h were fixed with formaldehyde and permeabilized with 1% triton X-100. The cells were stained with 10 µg/ml anti-tMeK-FITC antibody with and without the presence of free tMeK (10 µg/ml). The image was captured with the Zeiss fluorescence microscope equipped with Northern Eclipse imaging software with an exposure time of 100 ms. The picture shows the representative images of the staining.Page 5 of 8(page number not for citation purposes)tMeK antibody is highly specific. silenced region of the genome [4].Proteome Science 2008, 6:2 methylation is one of the important post-transla-tional modifications. The role of the protein methylationin protein functions is not completely understood. Pro-teomic studies of the protein methylation pattern in dif-ferent cell cycle stages and disease states, in response togene expression regulation and drug treatment would beinteresting. Using the tMeK-specific antibody for Westernblot, the trimethylated protein patterns could be detectedand identified. Using immunoaffinity-capture techniques,trimethylated proteins could be specifically isolated bythis tMeK-specific antibody and further identified throughpeptide sequence analysis, Western blot or ELISA screen-ing, microarray screening and mass spectrometry databasescreening. Presumably, this antibody can also selectivelycapture the trimethylated peptide fragments as a result ofthe protease digestion of purified proteins. Therefore, thiscould facilitate the proteomic survey of the trimethylatedproteins and identify the specific site of trimethylation.Similar approach has been reported using acetyllysine-specific antibody to identify the acetylated proteins in thecell through the immunoaffinity-capture technique andMS analysis [15].In summary, CH3I is a good choice for chemical trimeth-ylation of proteins as such immunogen yields more anti-bodies against tMeK than the antibodies against mMeKand dMeK. Single molecule of methylated lysine could beused as immobilized affinity ligand to selectively purifythe specific population of the polyclonal antibody againsteach specific form of Nε-methyllysine. The tMeK specificantibody prepared using this method is proven to be use-ful for detection, identification, isolation and localizationof the Nε-trimethylated proteins.MethodsMethylation of KLHAll reagents except mentioned were purchased fromSigma-Aldrich (St. Louis, MO). 25 mg of KLH was dis-solved in 10 ml of 0.5 M Na2HPO4, then 10 µl of theiodomethane solution was added every 30 min at 35°Cfor 2 h. The reaction was incubated at 35°C for another 4h. Saturated Na2CO3 was added to maintain the pH of thereaction greater than pH 9. The reaction was stopped with50 µl of the 2-mercaptoethanol at room temperature for30 min. The methylated KLH was then dialyzed againstPBS 4 times at 2-h intervals. After dialysis, the total pro-tein concentration was determined using the bicin-choninic acid kit (Sigma) as described by the supplier.ImmunizationThe methylated KLH (0.5 mg/rabbit) was immunized tothree rabbits (New Zealand species) with complete Fre-und adjuvant. The rabbits were boost with 0.25 mg meth-respectively. The rabbits were bled for serum preparation60 days after the initial immunization.ELISA5 mg of mMeK, dMeK and tMeK were dissolved in 1 ml of0.1 M Na2CO3. 5 µl of 70% glutaraldehyde was added atroom temperature for 1 min; then all the reaction mixturewas transferred to 10 ml of BSA (2 mg/ml in 0.1 MNa2CO3) in a 50 ml centrifuge tube. The reaction wasincubated at 35°C for 4 h then cooled to 4°C. 10 mg ofsolid sodium cyanoborohydride was added to each reac-tion. Each reaction mixture was then dialyzed against 500ml of PBS for four times at 2-h interval. The conjugateswere stored in aliqouts at 4°C before use.mMeK, dMeK and tMeK-BSA conjugates were diluted to20 µg/ml in 0.1 M Na2CO3. The ELISA plate was coatedwith 100 µl of methylated BSA conjugates in each welland was incubated at room temperature for overnight.The plate was washed with PBST (PBS containing 0.04%Tween 20) and blocked with 100 µl/well of 2.5% BSA atroom temperature for 2 h. The plate was washed withPBST for three times.The serum was titrated from 1:1,000 in each methyllysine-BSA plate in 2-fold serial dilution. The purified anti-tMeKantibody was titrated in plates coated with mMeK-, dMeK-and tMeK-BSA. Samples were run in triplicates. The start-ing concentration of the anti-tMeK is 250 ng/ml in PBSTcontaining 1% BSA and was diluted in a 2-fold serial dilu-tion. The primary antibody was incubated for 60 min andwashed with PBST for 4 times. Then, anti-rabbit IgG HRPconjugates (0.25 µg/ml) in PBST containing 1% BSA wereadded and the reaction was incubated at room tempera-ture for 30 min. After washing with PBST for 4 times, 100µl/well of HRP substrate TMB was added for color devel-opment. The color reaction was stopped in 20 min with20 µl of 1N HCl and absorbance at OD450 nm was meas-ured.Affinity purification of Anti-tMeK antibody10 ml of agarose beads (Sepharose CL4B) was suspendedin 25 ml of 0.1 M acetate buffer (pH 6.5), and 200 mg ofthe sodium periodate was added at 35°C for 2 h to oxidizethe agarose to yield aldehyde functional groups. Thebeads were washed with the 400 ml of the acetate buffer(pH4.5) then with 100 ml of distilled water. 5 mg ofmMeK and dMeK and 15 mg of tMeK were dissolved in 1ml of 0.1 M Na2CO3. The coupling reaction was per-formed by adding mMeK, dMeK and tMek, respectively,into the oxidized agarose (W/V = 1/2) at 35°C for 2 h thencool to 4°C. Then, 100 mg of the solid sodiumcyanoborohydride was added to each reaction and incu-Page 6 of 8(page number not for citation purposes)lylated KLH each with incomplete Freund adjuvant in 14days, 35, and 56 days after the initial immunization,bated overnight. The beads were washed sequentially with100 ml of distilled water and stored at 4°C.Proteome Science 2008, 6:2 scheme for the affinity purification of tMeK-specificantibody was illustrated in Figure 1. 10 ml of mMeK and10 ml of dMeK immobilized agarose (20 ml in total) werepacked into one glass column (2.5 × 10 cm) and 30 ml ofthe tMeK agarose into another one glass column (2.5 × 10cm). The column was washed with 200 ml PBST then 10ml control blank rabbit serum was allowed to passthrough each column and washed with 100 ml of PBSTand 50 ml of the 3% acetic acid. The columns were thenequilibrated with 50 ml of PBS.200 ml of the immune serum was initially passed throughthe 20 ml mMeK/dMeK column at a flow rate of 5 ml/min. The column was initially washed with 200 ml ofPBST then with 50 ml of 2 M NaCl. The antibody waseluted from the column with 3% acetic acid and collectedon ice. After 5 ml void volume, 25 ml of eluent was col-lected and 3 g of ammonia sulfate was added per 10 ml ofeluent to precipitate the eluted antibody. The solution wascontinuously stirred until all ammonia sulfate was dis-solved. The solution was placed at 4°C overnight and theantibody was precipitated by centrifugation. The columnwas washed with PBST again then equilibrated with PBS.The flow through was then passed through the same col-umn again until all the mMeK/dMeK specific antibodieswere removed by the column.The mMeK/dMeK column adsorbed flow through wasthen passed through a 30 ml tMeK column at a flow rateof 5 ml/min. The column was initially washed with 250ml of PBST then with 100 ml of 2 M NaCl. The boundantibody was eluted with 50 ml 3% acetic acid. After 8 mlvoid volume, 35 ml of eluent was collected, and 3 g ofammonia sulfate/10 ml of eluent was added immediatelyto precipitate the eluted antibody. The tube was incubatedat 4°C overnight and the antibody was precipitated bycentrifugation.The precipitated antibody is dialyzed against PBS and sub-jected to SDS-PAGE characterization and protein determi-nation using human IgG as a standard.Antibody conjugation20 mg of HRP was dissolved in 4 ml of citrate buffer (pH6.5) and 5 mg of sodium periodate was added and incu-bated at 35°C for 30 min. The reaction was stopped bypassing the mixture through a 15 ml G25 Sephadex col-umn. The brown color fraction of HRP was collected andstored at 4°C.2 mg of the purified antibody was added in 0.5 ml of 0.1M Na2CO3 and mixed with 1 mg of the activated HRP at35°C for 15 min then cooled to 4°C. 1 mg of sodiumthrough 15 ml of G25 Sephadex column equilibrated withPBS. The brown HRP fraction was collected and wasstored in presence of 50% glycerol and 10 mg/ml BSA -20°C.2 mg of the purified antibody was added in 0.5 ml of 0.1M Na2CO3. 1 mg of the FITC was dissolved in 50 µl ofDMSO. 10 µl of the dissolved FITC was added to the anti-body solution, vortexed and incubated at room tempera-ture for 30 min. The reaction was stopped by adding 10 µlof saturated Tris and then passed though a 15 ml G25Sephadex column equilibrated with PBS. The yellowFITC-labeled antibody fraction was collected and storedin presence of 50% glycerol and 10 mg/ml BSA at -20°C.Cell cultureMelanoma MMRU cells, a gift of Dr. R. Byers, Boston Uni-versity, were cultured with Dulbecco's Modified Eagle'sMedium (Gibco) supplemented with 10% fetal bovineserum (Hyclone), 100 units/ml penicillin and 100 µg/mlstreptomycin in 5% CO2 atmosphere at 37°C. For trichos-tatin A (TSA) treatment, MMRU cells were treated with200 ng/ml TSA for 16 h.ImmunoprecipitationA mouse spleen was crunched and homogenized withlysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl,0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodiumdeoxycholate, 100 µg/ml phenylmethylsulphonyl fluo-ride, 1× protease inhibitor), and the lysate was centrifugedat 12,000 × g for 5 min. The supernatant was used for bothIP and western blot. 100 µl of Protein A was packed in amini-column and anti-tMeK antibody (200 µg) in PBSwas pre-immobilized to the Protein A beads by passingthe antibody through the Protein A bead column 4 timesand washed with PBST. The crude lysate of the mousespleen (5 mg) was passed through the anti-tMeK pre-fixedProtein A beads once, washed with PBST extensively,eluted with 200 µl of hot 2× SDS-loading buffer and theeluent was collected.Western blotWhole cell lysate was prepared from MMRU cells by lyz-ing the cells in lysis buffer and sonicated. Cell debris wasremoved by centrifugation at 12,000 × g for 10 min andthe supernatant was recovered. Protein concentration wasquantified by Protein Assay (Bio-Rad, Hercules, CA). 50µg of proteins and 20 µl of IP sample were loaded for SDS-PAGE, transferred to polyvinylidene difluoride mem-brane, blocked in 5% BSA in PBST and probed with HRP-conjugated anti-tMeK antibody at 1:1,000 dilution withor without 10 µg/ml tMeK. Signals were developed by ECLsystem (Amersham, Piscataway, NJ) and visualized byPage 7 of 8(page number not for citation purposes)borohydride was added and incubated overnight. Sodiumborohydride was removed by passing the solutionautoradiography.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Proteome Science 2008, 6:2 stainingSubconfluent MMRU cells seeded in a 6-well plate with orwithout HDAC inhibitor treatment were fixed and perme-abilized with 1% formaldehyde and 0.5% Triton X-100for 10 min and blocked with 10 mg/ml BSA for 1 h atroom temperature. Cells were stained with FITC-conju-gated anti-tMeK antibody (10 µg/ml) with or without 10µg/ml tMeK for 1 h at room temperature. Photos weretaken with Zeiss fluorescence microscope equipped withNorthern Eclipse imaging software with an exposure timeof 100 ms.Competing interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsZL performed antibody production, purification andimmunoprecipitation. RPCW carried out tissue culture,immunofluorescent staining, immunoblotting, and man-uscript preparation. LHL and HJ participated in the anti-body preparation. HX and GL consolidated the studydesign, data analysis and finalized the manuscript. Allauthors have read and approved the final manuscript.AcknowledgementsThis research project is partially supported by Scientific Research, Experi-ment and Development (SRED) tax credit program of Canada and Guangxi Science Council of People's Republic of China.References1. 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