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Advantages Ad t and d disadvantages di d t off the horizontal g gel system y of gelcompany 2DGel flatbed 12.5% NF Large vs. traditional gel system Knut Büttner, Institute of Microbiology, gy Ernst-Moritz-Arndt-University y of Greifswald, Greifswald, Germany 2DGel flatbed 12 12.5% 5% NF Large for the FlatTop-Tower (gelcompany) • Pre-cast gels on non-fluorescent film-backing • Available pre-cast gels: – 2DGel flatbed NF 12.5% Large (200×255×0.65) – 2DGel flatbed NF 10 - 15% Large (200×255×0.65) (200×255×0 65) • • • • IPG-Slot 240 mm long Applicable for fluorescent staining (Lava Purple, Purple Flamingo) Fluorescent film-supported gels can also be stained with Coomassie Blue Also in small format (125×255×0.65) for 2×11cm IPGs FlatTop-Tower 4 Quelle: http://www.etcelpho.com Running conditions • Electrophoresis programme: Step Set [V] Set [mA] Set [W] Time [min] 1 100 7 2 30 2 200 15 5 30 3 300 30 5 10 4 1500 40 40 240 • Temperature: 15 °C Reduction and Alkylation Green: 15 min Equi A (with DTT), 15 min Equi B (with IAA) y 5 min in Equi q Buffer ((without DTT and IAA)) Red: Only The short running time allows to abandon reduction (with DTT) and alkylation (with IAA) of the proteins before SDS-PAGE. SDS PAGE Master Gel Image: 090317_B_subt_168 WT_Exp_BMM_Flat_40W_40mA_A_LP_450V Sample Gel Image: 090317_B_subt_168 WT_Exp_BMM_Flat_40W_40mA_D_LP_noEqui_450V Influence of the electric power on SDS-PAGE In 50 W gels protein spots become visible, which are superimposed by other spots in 2 W gels, or have not been stained. Master Gel Image: 081203_B_subt_168 WT_Log_BMM_500µg_Flat_4P_50W_1_480V Influence of the Electric Power on SDS-PAGE 2 W Gels 30 W Gels Dps, YphP0 50 W Gels YphP YphP Dps Dps YdaG, RplJ RplJ YdaG, RplJ RplJ YdaG, RplJ RplJ Reproducibility: IEF (traditional gel system) Some protein spots seem to be horizontally displaced in the 2D gel, when IPGs of different IEF runs are compared Reproducibility: IEF (FlatTop Tower 4) Master Gel Image: 081203_B_subt_168 WT_Log_BMM_500µg_Flat_4P_50W_1_480V Also comparing 2D gels with IPGs from the same IEF run shows horizontal displacements of some protein spots. Sample Gel Image: 081203_B_subt_168 WT_Log_BMM_500µg_Flat_4P_50W_2_480V Reproducibility: SDS PAGE (traditional gel system) In the traditional gel system there are often serious differences appearing i between b different diff runs. Reproducibility: SDS PAGE FlatTop-Tower 4 The commercial pre-cast gels from gelcompany offer very good reproducibility over long time periods. Master Gel Image: 081203_B_subt_168 WT_Log_BMM_500µg_Flat_4P_50W_1_480V Sample Gel Image: 090223_B_subt_168 WT E WT_Exp_BMM_Flat_40W_40mA_B_LP_200microns_1 BMM Fl t 40W 40 A B LP 200 i 1 Staining: LavaPurple vs. vs Flamingo Green: Flamingo-Staining Red: Lava Purple Purple-Staining Staining M t G Master Gell IImage: 090114 090114_B_subt_168 B bt 168 WT WT_Log_BMM_500µg_Flat_4P_50W_B_Flam_380V L BMM 500 Fl t 4P 50W B Fl 380V Lava Purple stains acidic, low molecular weight proteins less efficient than Flamingo, high molecular weight, basic proteins are stained better with Lava Purple than withFlamingo. Sample Gel Image: 081126_B_subt_168 WT_Log_BMM_500µg_pH 4-7_Flat_4P_50W_480V Staining: Lava Purple vs. vs Flamingo Master Gel Image: 090119_B_subt_168 WT_Log_BMM_500µg_Flat_40W50mA_A_Flam_430V Flamingo Staining Sample Gel Image: 081203_B_subt_168 WT_Log_BMM_500µg_Flat_4P_50W_2_480V Lava Purple Staining Flamingo often causes a black shade on the pre-cast gels. Staining: Colloidal Coomassie The pre-cast gels on non fluorescent film-backing (for Fluorescent staining) can also be stained with colloidal Coomassie. Sample Gel Image: 090526_NCF_10grad_1 Identification of Proteins Horizontal Gel Traditional Gel Identified Proteins (without duplicates) 885 Identified Proteins (without duplicates) 705 705 Number of identified spots 1548 Number of identified spots 1206 269 Sum of identified Proteins, which have not been identified in the horizontal gel 90 Additional not identified Spots 406 Additional not identified Spots 282 Number of Proteins with exactly two Spots 178 Number b off Proteins with i ih exactly two Spots 119 Portion of Proteins with exactly 2 Spots 12 % 12 % Portion of Proteins with i h exactly l 2 Spots 2S 10 % % Sum of identified Proteins, which have not been identified in the vertical gel Proteins, which have not been identified in the Vertical Gel Smc YfkN YfkN PolA (AlaS, PolA) LeuS (LeuS, PheT) YosN (NrdE, YosN) YosN (NrdE, YosN) RecN (ProS, RecN) LepA PhoD GroEL YtcJ YqjN YqeV (YqeV, IlvD, GcvPB) YfmG GabD (PdhD, GabD) YdbR (YuiE, YdbR, SerA) TuaD SpsB GltB MalA, MenF (MalA, MenF, PdhD) MmsA ((MmsA, YfmT)) SpsB GntZ (YqjI, GntZ) HemAT (HemAT, MurAA) YabN ThdF YoaC (YoaC, Obg) LpdV (LpdV, ArgG) AccC (AccC, YbbT) ProA MurC PatA YclM YitA (Sat, YitA) OpuBA (OpuBA, PatA) AraR PurT (TufA, PurT) PatB Drm ArgF, YjcS (Asd, ArgF, YjcS) PepT YxeP (AspB, YxeP, YvlB) YyaF (LeuB, YyaF) Buk (Buk, AroA) YrvO (CitZ, YrvO) Drm YlxA PrfA (PrfA, FtsZ, Icd) YhfS (YhfS, AroA) DnaI YxzC (Mbl, YxzC) YoxA (BioB,YoxA) GutB (IlvC, GutB) HemE YtqA YkpB YcsN LacR (YfnG, LacR) GltC YkkE YlmD YerQ AcuB (YgxA, AcuB) YbgE RsbU AcoA (Hag, AcoA) YtbE RnhB (FabI, RnhB) EcsA YwfL YhfI NfrA YfnH YycF (CodY, PurC, YycF, YurL, Mtn) KduI (KduI, YaaD) YbaJ (FabI, YbaJ) TrmD GamA (GamA, FabL) EtfB YvzB ExoA (ExoA, YwtE) SdaAA RsbR YurL (Sat, YurL) DppA (MinD, DppA) DapF PstS Y H YngH AlsD (Sat, AlsD) YfjR (Rph, YfjR) YuxI (RplC, YuxI) RibE YqeU (SrfAD, YqeU) SpoVFB RsbX RsbX YvrE (PpaC, YvrE) YitL (YitL, Hag) YxbB YdbD GlcR NfrA YlnD (YjbV, YlnD, YkrS) YhxD (YhxD) PyrD (YaaD, PyrD, FabHA) FruK TrxB ThyA (ThyA, ResD) YwqL (YwqL, PurF) YusA (RpsC, YusA) EtfA YvzB YodE (FabHA, DapB, YodE) YviA, DnaX YtzF MinC (MinC, YpjG) YknY YfmJ, YosP (YfmJ, NrdF, YosP) YbgG YdfO (Hag, NadE, YdfO) YfnE YitH YdhC, YtdA (YlnE, YdhC, YtdA) YbfQ (YwaA, YbfQ) ThiL (MetK, ThiL) PksD YkfA YfnD YxaA (YdiR, YxaA) IspA HemB (MreB, HemB) YkfC AroD, SpsK YjkB (YlmE, YjkB) HisH YsiA PyrE UppS (UppS, SpsI) YdfN YhdZ YkvJ YwnB PhoP (PhoP, YlmA) YcnD YheG Hpr YqgN YsiB MsrA QcrA YcsE (Adk, YcsE) YabD (YabD, TenA) AroC (TenA, YwfI, AroC) YsnA (YsiB, YvbH, YsnA) YhdZ YpiB (YpiB, AroK, Xpt) YtqB PabA (PabA, HisA) YueJ YfiR (SodA, YfiR) YgaC (LuxS, YgaC) YvdT YxkC Tmk InfC (InfC, YpjQ, YceE, YceD, ) YvqK YdaF (YdaF, YwjH) YdgI YtaG YpjQ YrpE (YrpE, YwrO) YjcK YppQ YokJ YuaC YcnI (MreB, YcnI) MoaB YrdA (YrdA, RpsG) YezE (Efp, YezE) YlaL (YlaL, YwrO) YsmB, PheB YsnB FlgC MoaE YacN (YacN (YacN, YjoA) Ki C KinC YwqK (YwqK, Tpx) YncF YosS YncF, RpsI YtsP YxiF YdiB (YhcB, YdiB) AtpF YaaK (YaaK, Apt) YxbD YllB RplV (RplV, RpsQ) Def YxjI (YkrT, YxjI) YpdQ YrhF RsbT, RpsQ, YdcD YeeF RplO YhcB YwpF YusI YvbK YwqH RpoE FhuC SpsL (Nin, SpsL) AroH YusE FliT YozA (YozA, RplS) YjbI AtpC RplR (RplR, RpsM) YppE YrrK YqaE RpmA YmcA (YqhS, YmcA) FolB YopJ YhfF Veg, YerC YpjD YqhY NusB YjbL (YjbL, YjlC) RplK Abh RplS (YwbC, RplS) YwdI YunG YphP (Dps, YphP) YdcE Spo0F SpoVS YlxM, RpsQ (YlxM, RpsQ, RpsD) RpsJ (YpfD, RpsJ, RpsS) AtpF Crh (RpsS, Crh) YtpP YetH, RpsP, YorD YjcQ YusU YutD YozE YcnE YdbP (CspD, YdbP) ThiS YutI (YutI, CspD) YitW AtpF YkvS, YqfC YozE YlaN (YkzG, YlaN) YkuJ RpsM YaaA YwhB YfkK YpmA When e more o e tthan a o one ep proteins ote s have a e bee been identified de t ed in o one e spot, be before oe the brackets there are the proteins, which have exclusively been identified in the horizontal gel, within brackets there are all proteins, which have been additionally identified in this spot. Sample Gel Image: 090526_B_subt_168 WT_Exp_BMM_450µg_Flat_A_NCF_460V_100mic Created by Delta2D - http://www.decodon.com Proteins, which have not been identified in the Horizontal Gel CgeA MutS LonA LonA YdiF KatE YetO (YvgR, YetO, YkfD) YusJ (YitJ, YusJ) YokI (YokI, DhbE) YocI YvbJ YhcX (ThiC, YhcX) MenD YheB (AspS, SrfAB, SrfAA, YheB,) PucD XynB RocA (LeuA, RocA) YmfA YcgN BkdB PnbA (PnbA, PdhD) PbpE FliD B lH (B BglH (BglH, lH Y YugT) T) PbpE OppA LicH YaaH SpeA (SpeA, YfmT, YueK) FtsA YpwA YpwA SpoIVA NadB (AspS, NadB) FliY YrrO (YrrO, GatB) GlpK DnaC DacA (YqjI, DacA) MelA YsnF YqbM (FabF, YqbM) YxeQ YaaN (YhxA, YaaN) PurK (PurK, YqiG) YloI Dxr (Dxr, YbaL) SpsE RapG YugK YacK DegS (DegS, YydG) DnaN (SucC, DnaN) AlaT (HisZ, AlaT) CitA (YjcJ, CitA) RbsK (NusA, RbsK) ManR (IolS (IolS, ManR) HprK AnsA YabC YloC (Mdh, YloC) FabHB Gcp SpeE YvcK (SucD, YvcK, YfnG) GlcK (GlcK, RpsB) YclP YfmF YcdF YfiY YojG YtbQ (YtbQ, YwfI) YwfC YwqC (TufA, YwqC) YvrH ((YvrH,, TenA)) YtkL YkrY CssR YqiX HxlA YdfQ YcgM HisB (YwjH, HisB) YqeK (AroK, YqeK) ClpQ (YwiB, ClpQ) RplM GsiB RpsE (RpsG, RpsE) YsmA YwkD When e more o e tthan a o one ep proteins ote s have a e bee been identified de t ed in o one e spot, be before oe the brackets there are the proteins, which have exclusively been identified in the vertical gel, within brackets there are all proteins, which have been additionally identified in this spot. Mastergel Flat Top Tower (Horizontal Gel) GltA GltA GltA Smc PycA PycA PycA PycA SrfAC SrfAA SrfAA SrfAC OdhA OdhA YfkN YfkN RpoB IleS PyrAB PyrAB RpoB PyrAB RpoB CarB Ald IleS CitB SrfAA CitB CitB SrfAA, SrfAB ValS SrfAA CarB AlaS,PolA SecA SrfAA MetE PcrA NrdE NrdE,YosN GidA GidA MetE ThrS SdhA,YkqC DhbE DhbE DhbE YkqC KatA,YtcI SdhA SdhA PyrG YvfW YhfT YvgQ YvfW GuaB GuaB GuaB SerA,FabL GuaB, LeuC SerA IlvD YtcJ Pyk Pyk ProS,RecN PdhC SrfAA GlmS,AdeC GcvPB PurF TuaD YqeH,BioI,Zwf YqeH YutJ GoxB,MurAB GoxB,YutJ PdhA PdhA IlvA,GlyA OppD PatA AraR YjcJ GuaC PfkA MetA Prs, LeuC SerA PheS,Ald YjcI YxjG YxjG YlmD Prs Prs CcpC AccD,SpoVFA ArgC IlvC,IolS HemE MetK,QueA YvcT YbgE CysK, YfjP AccD,RpsB,CysK YqfL RpsB YvfV YjbN SucD CysK LeuA YlnE YlnE AccD SucD YgxA,AcuB PurR LeuA, PheA PyrK FabI PurR YlnE,YdhC,YtdA YqiK BioD NfrA YbxB YjbV NfrA CysH FabI, YqfN YfnH RsbX RpsD,AroA SpoVFB Maf Upp,RpsD SpoVC Dgk, RpsD, Upp Pgk Dck,YdiC YdfN YkrB Udk YceC Pyk, YhfR, SigH YwjH,YodD,YueJ YwjH YhfK YqaP CspR YtqB RpsG YwrO YvqK YjcG YdaF,YwjH NusG YrkL YjcK YaaE ClpP RpsB YwiB KinC YlmF YlaL,YwrO YuaC YwjB Ssb FlgC AroA Yj A YjoA YjoA RpsI YrhF YugI,RplS YwpF YncF,YosS RpsG, IleS YtsP Def YhcB,YdiB AlaR,YkvM RpsG AtpF YaaK,Apt YhcB YkrT,YxjI YxbD YvcI YkrZ RplO Nin,SpsL GroEL Ndk GsiB YeeF YlxS IlvC YurV YvyD AroH TufA AroA YcsD FliT AtpC RplR,RpsM YqhS,YmcA YkrT,YviF Dps,YphP YjlC YrzC AroA FliS YrrK RpoE YqiW YklA YjbI YppE YneT YutE RibH,YdeM AroA YozA,RplS Dps,YkrZ Dps YslB,YqiW Ndk SpoVG YkrZ YvbK YwqH MetS, PnpA RibH YabA YqaP YwlF YfmP YusE RsbW YwqK,Tpx Tpx YkvM YhgC YllB TufA PurE RibT, Ndk TagD YusI AroA YciC Tig Apt YokL, RpsG TufA Tpx RpsB PpiB IlvC TufA YraA,Ssb MoaE YdaG YxnB PpiB, YwlF PpiB YraA,GroEL YkvM YxiF AhrC RibT GreA Tig DivIVA YesJ, DivIVA YjoA YwlF PurE MntR,YgxA Ndk YqeY YkrZ YkrZ ClpP RplJ YwnH Y N Yj A YacN,YjoA MntR YpdQ YkrZ CheW,InfC LysC YraA DivIVA RpsG YjoA,PpiB TufA,YwiB DnaK YokJ LysC,CheW MreB,YcnI Ssb YutG DivIVA YtxH RplJ TufA YsnB YugI RplV,RpsQ InfC, YpjQ, YceE, YceD, Efp Efp,YezE SodA, YaaE InfC,AhpC YtoQ,RpsG RpsG AhpC AhpC HprT, InfC YpjQ HprT ClpY Efp YtaG YtoQ,TufA YceD,YceE,AhpC YceE,YceD GrpE YdgI Tmk SodA,YaaE YocJ,YrdC BsaA,YkrW RsbT,RpsQ,YdcD YceE, YceD NudF LeuD,RpsD MoaB YjcF TufA Tig AccB,RpsD AccB,YceE,YceD DivIVA YlbI SerA YqzC Hit MreB,FlgE YkrT YuaI BsaA YtoQ YwpB YaaQ RpsC AccB YmaB TufA YvdD Fur YrdA,RpsG YppQ RplK,YsmB HisG HisG YkuU,YkrT LeuD YtpR,YceD,RpsD,YceE YpqE DfrA BsaA RplC, RpsC Adk,YcsE RpsD SodA,YaaE YdjL YwjH Ctc YkrA,HisG,HisI YhcW,YkrX TufA, YfkM TufA PabA,HisA SodA,YfiR TufA SerA,YgaF LeuB Eno GrpE ArgJ,YceD YsaA,HisI Gmk,GtaB TufA RpsD, TufA YocJ ClpP YhfK YrpE,YwrO YkrT YdfG ThiG,YurY YqgX Dra,GtaB PyrR YkaA YodC,AroA RimM YvdT YutF YkuQ TufA YurY HisA LysC,YkaA DeoD TufA YkrT YwjH YjcG RpsG YaaD,TrxB PyrR TufA,LuxS Frr LuxS YxkC YvaB YlbN RplC,YuxI Gmk,RpsC YsiB,YvbH,YsnA YsiB FusA CysC YhfK LuxS,YgaC TufA RplC,YwfI,TenA TenA YsnA ComA TpiA YoxD RpsD,BioW YwjH YwjH RpsB TufA YsaA,GtaB Cmk CysC, Cmk PyrR YvyD YodC YoxD YueJ YjcG Tig RpoA GtaB PurQ YutF YabD,TenA ThiE PyrH YhfR Hpr Pyk YwjH YqgN YgaF CysC LeuA Frr YceC,AroA PurN MsrA AroK FolD YvyD YoxD RpsD SerA YkqB,Rpe YtkD YeeI TufA TufA RpoA,YfmS TufA YxeH,Sat ThiG,YkuQ RplC,ComA Soj TenA PdhA YlpC PyrE Xpt YgaF QcrA YeeI TufA TufA,YxeH Sat,AlsD YurY YkvJ Rpe,Pgk AroK YkrT YfmS RsbR FlgE YwnB TufA KipI TpiA TpiA CodY,PurC TenA YvbY YhdZ YfmS,YdjL YkrT Sat,YurL PurC YwfI, TenA TenA,YwfI,AroC PstS YfmS YkoB SdaAA PunA PurC Rph,YfjR TenA, YvrH RpsB Spo0M Spo0M Hag,YvzB HemL TenA Hag DapF ThrB ExoA,YwtE YkoB TasA YshC,YvaK PhoP,YlmA MinD,DppA YhdZ FlgL Hag TufA SpeB YkrT YwtE CodY,PurC,YycF,YurL,Mtn KduI,YaaD YvaK YcdH RpsD YheG YuaE YlpC,PurB YvaK, YkrU FolD YktC YcnD SigH RpsD, Pgk, HisH, Upp HisH PurN YpiB, AroK, Xpt YuaE DhbA DegU,YlmE YdiC,YceC YrxA YsiA YtiB YddK YktC YwfC YngH YwqG EtfB YvzB YxbB YtbE YwqG PpaC PpaC,YvrE Mdh ThrB MetE GtaB HisF YwfC YvbY FliG MreB,YwaA YitL,Hag SpeB HisF YkwC PanB SrfAD,LexA PdhB,TufA CheV BkdAB,FusA Mdh,Icd GlyQ,DapA YacC PyrH YjjA YkwC YqeU SrfAD,YqeU SucD,RplC FabD,PdhB YhxD PpaC YfnF YojN,Dat GlyQ FruK Tsf YdbD YokG YokG YceD, YceE YdjE EtfA Mdh,Hag YaaD Tsf SigD HisF DhbA,PdhA,CysH DhbA YcsF DegU YfkO OdhB Dat Tsf GtaB YaaD,TrxB YceD, YceE TufA TufA,PdhB PanC YcdH YaaD ThiD,DapA,RocF FbaA Tsf YkrU,FbaA TrmD AroA DhbA UppS,SpsI YfnH YcsF YlmE,YjkB DegU NagB,YjdA YdiC,LeuA RibE Upp DhbA YhfI YitV DegU RpsC,YusA YjbK FabI, YbaJ PksC,FabI ResD PspA GamA,FabL PyrF YufM YktB YkoF PdhB,TufA Pta YtqI,YhfP Hag,YvzB Mdh TrxB Tsf,YaaD FbaA AcoB TufA NusA Hag BkdAB Hag,FusA,Mdh YsgA MenB YddK YdiR,YxaA IspA NusA YfmJ,NrdF,YosP FabHA,DapB,YodE FbaA,YvgN NadC, YsgA SerA,GapA PurM,RpoA GalE TufA Hag YvzB Hag, Tsf DhbB LeuB BkdAA Hag,YvzB MenB YjjA Y fL YwfL SpsA NusA GalE RsbU MenB NadC NadC,YjjA MenB,YtpQ MenB,FolD YusV FabI ArgG GroEL Tsf YaaD NadC MenB,YtpQ MenB AtpG EcsA FabL YtmQ CysK YviA,DnaX FabI,RnhB YflG Yi S YitS GlcR FolD,YtzF PurH FabI YwqL,PurF YjbV,YlnD,YkrS YknY FabL FolD YtzF PyrK ThyA,ResD PdhD YvyE YvfVYitH Spo0A YtrE YfhB,YqiD Sul YitH,YlnE CwlC ArgB YcbG FusA FolD PheA YjcH MinC,YpjG SucC SucC, YbfQ YwaA,YbfQ GapA Hag,NadE,YdfO Tsf YaaD,PyrD,FabHA SucD, YqfL PurM PurM DhbF, YdjL PurM YbgG LeuA,MenB,DhbE MetK PheA,PurR RpoA RpoA DhbB YdjI MetK, YvcT GroEL YxjG,YxjH LeuB YhdN,SucC,GapA HemH,Pta YwjI Tsf,YaaD Pgk YqjE YqjE GroEL PepT RpoA InfB Hag,AcoA NadE FtsY YwaA Eno PurD YxbC YwjI YcsN PksD YqfS ClpX YqhT SucC YxjG,HisC,YxjH YkrS, GapA YulF FtsY YqkF IlvC, YjlD, IolS YkfA YfnE YisK MetK,SpsD YwaA YumC IlvC,Cah,YkcA YfnD YdjL,SucC YdjL SucC YwaA Eno,YkuI Eno Eno LeuB LeuB,YyaF RecA YkrS,SucC HisC YumC MreB,HemB IolS IolS RpoC YfnG,LacR YtqA YbbI SigB YerQ MetK YqhM YkkE GltC KsgA RpsB,AccD SpsD YciC YpfD YpfD PurD,LysC,TufA PurD,TufA Pgk,Icd YdjL,YkrT YxjG,YhdR,YkrT YjcI YkrS YxjH,Hag YkrS YumC YpfD TufA TufA TufA YkrT YkgB,YvaA GapA AroA,GapA IlvC,GutB CcpA IlvC,TyrA YurP,Mbl Fmt,PyrB CcpC RpsB,CcpC FusA, YpfD YbbT TufA TufA,PurT Icd FtsZ YkvY AroA YcgT Prs YfhM RpsB AroD,SpsK YkrT,YkrV Drm Asd,ArgF,YjcS YhdR YhfS,AroA YbaL IlvC,YoaD YurP PyrB,BioB YfnG Prs Bcd RecA,ThrC AroA YurP IlvC YyaA GuaC,YqjK Prs YkpB RpsB YpiA,AroF AtpD FusA FusA YciC GlnA PrfA,FtsZ,Icd Drm YtsJ DhbC AckA,DhbC,YvaA RapF Sat CitZ,YsdC,SerC YkrS IlvC BioB,IlvC YeaC YyaA YkfC RpsB RpsB AtpD YbbT ClpX,ThdF YmfH FabF GlnA LysA YtsJ DhbC HisZ YugJ CitZ IlvC,YtxK Buk,AroA BioB,YoxA ThiF,Mbl YeaC Mbl,YxzC YocD DnaI LysA HemL PyrC DhbC AckA,Ald YqiG SerC PlsX Mbl CggR Fmt MetA,AroA,YlxA Prs Csd DhbC PurH AspB,YxeP,YvlB YtoP YtoP PyrAA YqfP,YhaM ExuR YhaM AccA AroF, ThiC PdhA, YdbM YhfJ YpdA,PfkA GroEL AtpD AtpD YtjP YbbT OdhB,ArgH Pgi Pgi,MetK PurH,DhbC MlpA YwfG AspB IlvC,YvlB CitZ,YrvO PdhA YhfJ YjlD PfkA,AccA YlxA Tig YuiE AhpF SigA,YtjP ClpX YclM YlmB,AmpS MetK,PyrC YkrW PatB SerC YsdC SerA PhoH AccA PfkA YhaQ AhpF,SigA YabN AccC,YbbT GlnA,MleA OpuAA,HisS TyrS,DhbC HisS Csd AspB AspB YvyH YsdC,GlyA MurB GltB,FlgK GltB FusA AtpD ThdF HisD,ClpX HisD ArgH,YmfH Pgi ProA CinA,Sat YkrW,Sat AspB NadA SerA YjcJ YdbM MurC GlyA YhaA,GlyA,Sat Sat,YhaA GsaB YjcJ PyrAA YjlD YceH YsdC MoeA,AmhX YjlD YjlD PdhD OdhB,GatB ArgH Obg MleA GroEL,YdbR GroEL BglA,PdhD,GuaA AhpF, ThdF MalA,MenF,PdhD PrfB,GlnA Sat GsaB OpuBA, PatA PdhA ArgD ArgD YurX AtpA LpdV,ArgG GroEL GroEL GroEL GroEL,DnaK YuiE,YdbR,SerA YoaC,Obg ArgG,YqjI Tig YloV GroEL GuaA,PdhD PdhD PdhD GatB YurX AtpA GatA HisS GuaA PdhD,GabD PdhC MetK SpsB GatA YqjI,GntZ HisS BioA YqjI Sat RibA,YhxA NadA YceH GcvT Sat,YitA YjlD YphC BioA Tig GroEL GroEL PtsI GlmS PdhD PdhD Hom,PdhC DhaS,YfmT,YueK AtpA SerS PabB PurA MurF PurA,MurF HemAT,MurAA GlyA IlvA GroEL LepA PdhC PdhC YurU Hom GudB DapG OppD Tgt,YutJ YjlD GlyA, CitG SerS YxeK YhxA GudB,GudB1 YhfE,AroF PhoD YlaG PtsI DnaK YurU DltA DhaS,AtpA,YurX YurX MmsA,YfmT PurA, MurF, CitG LicH CitG OdhB DapG PtsI, GroEL DnaK,YvgR DnaK GlmS PdhC,YurU YlaG, DltA, YurU YkpA,LysS PurH,YueK CysS,PurH PurH PurH AsnS,YueK GcaD PurA MurD MurD P B PurB BioI YutJ LysS,GltX YurU YueK,CysS SpsB ClpY MurD,YvfW OdhB, Gid PurA, MurD AroF,YhfE LeuC BioI LeuA Gid AroF AroF FliI YjlD YkpA, SrfAA GltX PurH AsnS YopQ ThiC AspS AspS GlmS SrfAA, SrfAB, DnaK, AspS, ArgS Pgm,PckA YfmG Zwf, AsnS PurH LeuA AspS MetE SrfAA MetE YqjN YlaG Zwf GntK, SerA AnsB,GcaD Pgm YllA YqeV,IlvD,GcvPB LeuA GcaD SerA YjbG ThiC, DnaK DnaK AspS, DnaK PnpA IlvC SerA LeuA PurF PurF PurF DnaK Pyk,YhxB ProS FabHA IlvB ThrS CheA SrfAA, SrfAB YvgR DnaK,YvgR Pyk,YhxB IlvD Pgm SerA YvgQ GuaB PurF DnaA FusA SrfAB,SrfAA HtpG HtpG HtpG Tkt,YhxB YvgQ YvfW GuaB PurL SrfAA, SrfAB FusA FusA Hag Tkt Tkt Pyk IlvB LigA InfB PnpA PyrG IlvB LigA MetE, PnpA MetE PnpA YkoD,YdaP GuaB LeuC AroE RpsB PnpA YfmR PyrG PyrG PurL PurL PurL PurL FusA FusA MetS PnpA YjbG FusA YlaG ProS YshC YvgQ MetS GlyS GlyS Vpr YjbG YitJ,PyrG ThiC AroE,TufA GpsA GlyS GlyS YitJ IlvB KatA OpuCA ThrS ThiC ThiC ThiC YitJ,ThiC SdhA AcsA DhbE DhbE YkqC ThrS ThiC ThiC,YitJ FusA MetE MetE ThrS ThrS,Fbp ThiC ThiC, IlvC ThiC SrfAB, SrfAA PurL YlaG YlaG PrkA ThiC AsnB SrfAB, SrfAA, PurL, FusA FusA YlaG YlaG PrkA ThrZ AsnB YsmB,PheB LeuS,PheT MetE GidA ThrZ RsbX MetE MetE SrfAC YlaG SrfAA GyrB CitZ,NrdE GidA MetE RpoB, YrrC Pps NrdE,YosN TagE SrfAB, SrfAA PheT,LeuS ClpC ClpC SrfAB, SrfAA LeuS AlaS Ald SrfAB, SrfAA DhbF ValS ValS AlaS ValS SecA SrfAA,SrfAB CarB DhbF SecA SecA SrfAA,SrfAB PyrAB CitB SrfAA RpoC SrfAA,SrfAB SrfAA,SrfAB SrfAC IleS AroA OdhA RpoB SrfAB PycA IleS, SrfAB OdhA YwbC,RplS YqaE AbrB YueH MgsA FliS YpsB YugI YopJ YwdI YdcN,YqhS YunG AbrB YviF,YxxG YkzA YqhY YhfF RplV FolB NusB AroA AbrB RplL RpsS,SpoVG SpoIIAA RplU CspD,RplL Veg,YerC YabJ SpoVG YpjD YlbA RplK YlxM,RpsQ,RpsD YabJ YjbL,YjlC YdcE YpfD,RpsJ,RpsS YukE YheA Abh Spo0F SpoVS PtsH AbrB AtpF RpsS,RplL RpsS,Crh Icd PurH YusU RsbV MetK CheY YetH,RpsP,YorD CspD,YdbP RsbV TrxA YtpP RpsF YjcQ YutD AroA YjlC PurS TrxA YutI YjlC YozE RpsD GtaB YcnE RplR YloH PurS RpsF YkzG,YlaN ThiS GroES YutI,CspD YitW PurH RpoA CspB CspB RplD AtpF YkvS,YqfC RplD CspC YozE CspC YkuJ RpsM YaaA YwhB YfkK YpmA Master Gel Image: 090526_B_subt_168 WT_Exp_BMM_450µg_Flat_A_NCF_460V_100mic Created by Delta2D - http://www.decodon.com Master Gel Traditional System (Vertical Gel) TufA GltA GltA Ald, DhbC GltA RpoB, GltA, SrfAC MetE SrfAC SrfAC SrfAC SrfAA, SrfAB Pyk MetE RpoB Pyk SrfAA SrfAA RpoB SrfAA, SrfAB RpoB SrfAA SrfAB SrfAA, RpoB RpoB PycA PycA SrfAA, SrfAB PycA, GltA IleS IleS IleS SdhA OdhA OdhA OdhA OdhA SrfAB, SrfAA SrfAB SrfAB PycA PyrAB PurL, SrfAA, SrfAB SrfAB PnpA NrdE Fbp, ThiC ThiC ThiC, YitJ GlyQ ProS MetE HtpG DnaK HtpG HtpG YvgR, YetO, YkfD Tig YvgR, YetO PucD AspS SrfAA Pgm, ThiC ThiC IlvD IlvD IlvD PdhC YurU YurU AsnS PurH IlvD GntK LeuA LeuA OppA LeuA PurH PckA, ArgS Sat, NadA RibA, AmhX NadA GlyA, BioA GudB, GudB1 PurB AspB PdhA Icd IlvC YurP, IlvC AroA YdbM YjlD PyrAA YurP AroA, IlvC ArgD YkuI YumC SucC, DnaN SucC SucC LeuB NusA, RbsK YqhT BkdAA AcoB YwaA DhbB HemH Hag Hag PdhB YdjE PanC YtqI, YhfP, NusA, Hag CcpA NrdF DhbB ,FliG GalE YwjI Hag, YtqI YtqI GapA GapA YeaC Hag YwqG Hag, TufA YdjI, Tsf YabC FabHA YbbI ExuR IolS, ManR HprK Mdh NadE YqkF Cah Gcp Dat Dat BkdAB NadC YisK FabD AnsA YokG Hag, PpaC PpaC Mdh, YloC FusA YqfS Fmt FabD Mdh NadE Tsf, GapA DapB SpeE YcgT GuaC, PfkA Mdh YcdH YvcT YwaA SigB FabHB YyaA AccA YqfP PdhB YwaA, Gcp IolS YkcA ,IolS TyrA, YtxK PyrB YocD ,YhfJ GuaC, YqjK YdiR YhdN YwaA, MreB Hag BioB YhaM NusA DhbB MetK MetK, QueA YoaD TyrA, IlvC ThiF MetA TufA YwaA YulF ThrC, ArgC ThrC TyrA Mbl PfkA TufA PurM FtsY FtsY IlvC CggR YhfJ GroEL, TufA InfB,, YqjE qj SucC, YkrS IlvC GltA YyaA YxbC YugK YkrT, YjcI, SucC YkrS ThrC, RapF IlvC IlvC IlvC YjlD PhoH GpsA YkrS YugJ, AroA YtoP YjcJ, CitA ArgD, YjlD RpoA RpoA RpoA YqhT YkrS, YugJ ThrC, RapF YsdC YacK YjcJ GcvT YjlD YjlD Pgk, LeuB PurL YqjE RapG SpsE CitZ Eno Icd Icd ClpX, YqhT YhdR, YjcI HisZ, AlaT SerC PlsX Eno Eno YkrV YxjH YjcI Pgk Pgk YdjL Icd YkrT HisC GapA AckA Dxr, YbaL SerC CitZ PdhA FtsZ YkrT YxjG YxjG YkgB, YvaA YvlB CitZ ,YwfG YpfD Eno Asd ,RecA Bcd YxjG, HemL YkvY GapA YvyH YsdC YqeH ArgD YqiG Sat, PurH AspB YvlB RpoC, GsaB YtsJ, DegU, Asd PurK, YqiG BioA YhxA, YaaN GsaB YceH TufA YlmB YdjL TyrS, PheS CinA YkrW MoeA MurAB MurAB OppD YsnF YpfD RecA TyrS, DhbC, YvaA, GlnA, PheS YkrW PurB DapG, PurB DapG Tgt LeuB FusA LysA MlpA, AspB YhxA GudB YhfE GoxB DegS, YydG LysA PurH YpfD LeuB, LysC TufA FabF, YqbM YlmB YqjI YtsJ PyrC, DhbC, Ald DhbC, Ald YpiA PurD PurD PrfB Pgi YkrW AroF YutJ PurD Pgi YqjI PyrC, DhbC HisS, Csd AspB AspB YloI YqjI YciC DnaC TufA YmfH, GlnA BioA, HisS GlyA Sat Sat, GlyA YutJ YqjI, DacA YqjI SerA YbbT, TufA TufA ,YbbT TufA PrfB YlmB GlyA FliY YciC AtpD TufA TufA ArgH MleA GlyA YxeK YxeQ BioI YmfH, GlnA Obg AmpS Csd CitG YciC FusA AtpD AtpD TufA CitG, NadA SerA OdhB, FusA ArgG ArgG OpuAA MurAA, GlyA, YhaA YciC AhpF AtpD, GroEL GlnA YqjI LicH GlyA, CitG YvfW AhpF AhpF AspS, TufA YtjP OdhB PurA OdhB FlgK SigA TufA HisD, TufA HisD GatB GlnA GlnA, MetK MurD LeuC FliD YpwA FtsA IlvA NadA SpoIVA AtpD, YbbT GcaD, MurF IlvA PurF, GuaB LeuC BglA MetK, AtpA GcaD, YtcI YphC LeuC AspS, NadB GlpK AtpA AtpA MetK GatB YrrO, GatB AtpA AtpA Zwf, GcaD MurF, GcaD, PurA PnbA, PdhD SpeA, YfmT, YueK GatA Zwf PurA PurA PbpE PurH GatA GatA, YopQ GcaD MetK, YqjI, LysS MelA PurN YfmT SerS Zwf AnsB Gid YaaH BglH, YugT YueK SerS GcaD GcaD YvfW YpwA PdhD, Hom DhaS CysS DhaS, PurH, SpeA PabB Zwf LeuA PurF PurF PurF Gid AccA PdhD PbpE PurF YhfT GuaB DnaA YqeH Tig YmfA PdhD Hom Hom GltX BkdB ClpY GuaB FliI F A FusA GuaA, PdhD GuaA, PdhD YurX DltA SerA OpuCA InfB PdhC, OdhB YurX GltX YcgN SerA Tgt PdhC YurU ,YkpA LysS, ArgS CysS GroEL LeuA, RocA GuaB GroEL YuiE Tig YkpA ArgS IlvD IlvB PtsI AspS AspS, SrfAB, SrfAA, YheB, IlvB LysS YllA XynB GroEL YkqC Pgi YtcI GuaB MetE, GlmS Pgm, PyrG IlvB GcvPB SerA, YvgQ YvgQ, SerA YloV SrfAA, SrfAB AspS AspS IlvB IlvB YdaP ThiC YvgQ SerA SerA SerA, YkqC GroEL GlmS ThiC SerA, AcsA DhbE SerA YvgQ YkqC GlmS GlmS SrfAA Pgm SerA MenD DhbE DhbE DnaK AdeC YvbJ YhfK MetE Pgm YjbG, Pgm PyrG, Pgm YshC YocI YkqC DnaK YlaG MetE, YhxB ProS Asd Pgm YitJ, YusJ YkoD, PyrG ThiC, YhcX SrfAA, SrfAB DnaK, CheA YhxB YitJ YitJ YitJ SdhA AsnB KatA AroE FusA SrfAA, SrfAB YhxB AsnB SdhA Tig CheA FusA ThiC PrkA SerA SdhA LeuC LigA Pyk Pyk Vpr ThiC PrkA ThrZ ThrZ LeuC PnpA PnpA MetE, Pyk NrdE YokI, DhbE PurL PurL LigA Tkt Tkt Pyk PheT ProS GidA PurL SrfAB, SrfAA LigA Tkt ,Pyk YdiF Pyk MetE PrkA FusA YlaG, MetS GlyS PrkA NrdE MetE MetS MetS PnpA, YdiF GidA GuaB YlaG GlyS GlyS YfmR KatE NrdE YlaG, MetS YlaG YlaG, SrfAB FusA FusA MetE MetE MetE, PdhC MetE MetE SrfAB, YlaG SrfAB, AhpC ThrS ThrS DhbC RpoC TagE MetE MetE SrfAA, SrfAB SecA GyrB ThrS FusA MetE GlyA GyrB SrfAB, SrfAA SrfAB, SrfAA FusA PheT MetE MetE MetE SrfAB ClpC PcrA CarB SrfAC, FusA DhbF PheT PheT ,ValS MetE SrfAB Pps, SecA ClpC ClpC CgeA SrfAA, SrfAB PheT PheT, ValS MetE PcrA FusA PyrAB Pyk AlaS SrfAA InfB SecA GltA LonA CarB PheT, PyrAB CitB AlaS MutS MetE AlaS SrfAB, SrfAA SrfAB, SrfAA SecA, AroA SecA FusA RpoC RpoB CitB CitB OdhA PycA LonA SrfAB, SrfAA SrfAB, SrfAA PyrAB Pyk MetE ValS SrfAA, SrfAB FusA LeuB PycA OdhA Pta ThrB PpaC PpaC, TufA Pta PpaC, OpuAA NadC YpdA Pgk SucD YojN DapA, Tsf FbaA FbaA YeeI CheV, Pta YfnF Tsf NadC SucD BioW Prs RpsB Prs RpsB Prs YaaD SucD, YvcK, YfnG GlcK, RpsB RpsB NadC YfhB, NadC YvgN YfmF YclP YfhB, NadC ThiD, RocF YflG YacC Mtn SpeB GlyQ YqiD, NadC YfmS TufA GtaB YusV PurQ CysK CcpC YqhM PurH MenB YqfL, FolD PurR YvaK Soj Tsf MinD TpiA, YkoB YwtE YutF YutF TpiA YjjA TenA PurC Rph FolD CysK CysH KipI CodY YkwC YkwC FabI YojG TenA, YlmA MenB TenA YlmE, DegU AroA, PksC YqiK PunA YwfI HisI TufA Pgk CssR ThiE HisA YceE, YceD Adk YkrX YkuQ, TufA YcsF ArgJ YmaB YkrT Cmk YkrY LeuA, NagB YsaA YsaA Gmk SpsI OdhB RplC YsaA, YkuQ YkuQ YtkL YhfK PyrH Spo0A Tig TufA, YwqC YurY FlgE, ComA YvrH, TenA YvbY SigH, PanB YitV Adk GrpE YvbH YtrE NudF FolD BioD YfkO PspA FabL Dra PyrF YceC YhcW AroA Upp Xpt, Upp YqiX YhfR YdfI LeuD PdhA YbxB Udk DeoD CysC YvyD PyrF PurN YdiC, Dgk YceD, YceE, RpsC YkaA YkaA YvaB Xpt Dck YceD, YceE Xpt YkqB GrpE TufA RpsD PurN Maf HxlA LysC PyrR YoxD Rpe, RpsD YktB YqgX IlvC LeuA Upp YjbK, RpsC RpsC YtmQ HisG YurY YtbQ, YwfI FolD, YwfC YktC YlpC YjbV YjdA YufM ThiG YwfC LexA DhbA ArgG, DhbA FabI, LeuA ArgB GrpE ThiG ,YxeH Nap SpsA YfiY FabI, DegU PspA YvyE, FabI YjcH TufA FlgE TenA, PurC, CodY YjbN YvfV YpjG TufA YkrA MenB FabI YcbG YlbN Ctc TasA HisF, SrfAD, YaaD SrfAD YqfL YgxA CwlC YfmS TufA TufA GlyQ YddK, YjjA Sul PyrK, CysK FolD AccD YqhM YcdF YsgA MenB ResD AccD MetK, SpsD YlnE MenB, SigD, YkrU MenB, YtpQ PheA YfhM MurB Spo0M Mtn Tsf YurP SpoVFA, Prs YhaQ, RpsB RpsB YdjL YkrT Tsf SerA YkrU RpsB YfmS CheV, TufA YaaD YaaD KsgA AtpG RpsB YkuU YodC SpoVC, YwjH YkqB SodA, YaaE CysC, YodC ClpP SodA, YaaE SerA YkoF YocJ YwjH YrxA YkrB YdfQ YcgM YpqE Frr YwjH, HisB SodA AccB HprT YrdC YwjH YtpR ArgG ClpY YtiB LuxS AroK, YqeK YfkM TufA RimM Efp AccB Efp YodD CheW YvdD AhpC AhpC YgaF RplD YjcG SpoVC YgaF YuaE ArgJ YkrZ YtxH, YlmF AhpC, YkrZ LysC Dps YjcG MreB Tig YwjB YwrO YraA NusG YesJ GreA YutG YuaI Ssb Apt, YitS RsbW BsaA YqaP YhgC YhgC YwiB, ClpQ YtkD Dps AlaR DivIVA YdaG YlxS TufA, DfrA YrkL Fur CspR YaaQ RplJ Dps RplJ AtpD p YjcF AlaR RplM GsiB YkvM Tpx YwpB Nin YtoQ TufA RpsG, RpsE YrzC YwnH YqaP YwlF YcsD YviF YutE YvcI PurE YdfG YqzC YurV YsmA RibT PpiB YqiW YklA MntR YjoA AhrC, PurE YjlC YqhS YdeM YwbC YqeY YxxG RibH Ndk TufA YkzA YslB YwkD YugI FlgE Hit TagD PtsH YneT FliS RpoA p CspD YdcN RsbV MgsA RsbV SpoVG CspD RplL YukE YabJ YtxJ YheA YugI YlbA RpsS ,YkzG PurS PurS TrxA AbrB SpoVG YpsB GroES Master Gel Image: 090526_B_subt_168 WT_Exp_BMM_450µg_LP_1_470V_100mic_marker RpsF CheY RplD RplU CspB CspB YloH CspC Created by Delta2D - http://www.decodon.com Not identified Spots in Master Gel Flat Top Tower (Horizontal Gel) Master Gel Image: 090526_B_subt_168 WT_Exp_BMM_450µg_Flat_A_NCF_460V_100mic 406 Spots could not be identified Not identified Spots in Master Gel Traditional System (Vertical Gel) Master Gel Image: 090526_B_subt_168 WT_Exp_BMM_450µg_LP_1_470V_100mic_marker 282 Spots could not be identified Created by Delta2D - http://www.decodon.com Pros and Cons of the two Gel Systems Traditional T di i l Gel G l System S • Running time: ca. 16 h • Reproducibility ep oduc b ty difficult d cu t • Spots mostly homogeneous • Very labour intensive work (casting gels gels, cleaning) • Lower gel cost (excl. Labour) • Low molecular weight proteins are often blurred, MWMin = 7.7 kDa) FlatTop Fl T T Tower (H (Horizontal i l Gels) G l ) • Running time : ca. 5 h • Very e y good reproducibility ep oduc b ty • Spots have higher conc. front • Much reduced labour intensive • Higher cost, but ready to use • Low molecular weight proteins in sharp spots, MWMin= 6.5 kDa)
PERFORMANCE
EXCELLENCE
