[PDF] ECLウェスタンブロッティングガイドブック

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1 ECL WesternBlotting Guidebook ECL

2 They're EXCELLENT. Time after time. That Excellence you can count on. ECL 10 ECLHybond ECL ECL ECL AdvanceECL Plus

3 ECL Electrophoresis Blotting Labelling minive / SE cm2 minive SE 600 Ruby 16x16 cm 4 minive TE cm 4 TE 42 / TE 62 TE 70 / TE 77 Membranes Hybond-P PVDF Hybond-ECL cm4 710 cm16 TE 42 TE 62 TE cm, TE cm Rainbow Marker ECL DualVue Western Blotting Ma 7S-taggedEC 3 MultiProcessor ECL Western Blotting Molecular We HRP HRP ECL Advance ECL Plus ECL ECL Acce ECL Detection Kit ECL Phosphorylation ECL Glycoprotein

4 Detection Analysis ssories rkers ight Markers ECL X / Hyperfilm-ECL ECL Mini-Camera ECL 9374 mm ImageMaster 2D Platinum ImageScanner II Typhoon 9400 / 9410 / Trio RI1

5 CONTENTS ECL 2 1 ECL ECL Advance ECL Plus ECL ECL PlusECL Advance ECLECL Plus DABECL Plus ECL Mini-CameraHyperfilm ECL Mini-Camera Hyperfilm Typhoon 39 CCD 40 5ECL Plus Typhoon minive TE70 / TE MultiProcessor ECL Western Blottingmetallopeptidase D DIGE / ECL Plus Western Blotting References 85 ECL 86 ECL 88

6 ECL ECL

7 1.1 ECLEnhanced ChemiLuminescence Horseradish peroxidasehrp ECL Advance /ECL Plus ECL

8 1.2 cyclic diacylhydrazides (1, 2) HRPH 2 O 2 (3,4) ECL(2)HRP 1,000 ECL PlusHRP ECL AdvanceECL Plus ECL AdvanceECL PlusECL ECL ECL Advance ECL Plus ECL - - ECL Plus10 ECL g 1 : 100,000 1 : 500,000 ECL Advance Blocking Agent 1 : 20,000 1 : 10,000 1 : 200,000 1 : 15,000 5 %0.1 % PBS-T Hybond-P / Hybond-ECL Hybond-P Hybond-ECL X (Hyperfilm ECL) X (Hyperfilm ECL) X (Hyperfilm ECL) (ECL Mini-Camera) CCD (ECL Mini-Camera) CCD (ECL Mini-Camera) CCD Scanner ( Typhoon 9400, Trio, ) Storm860 3

9 ECL10 ECL7-9 ECL10,11 1 pghrp HRP DABdiaminobenzidine tetrahydrochloride 2

10 ECLECL Advance, ECL Plus, ECL ECL Advance 2.3 ECL Plus 2.4 ECL 6 7 8

11 2.1 SDS-PAGE minivese260 CBB) MultiProcessor PhastGel Blue R PlusOne Silver Staining Kit, Protein TE 70 / 77 ECL Blocking agent MultiProcessor HRP HRP- ECL AdvanceECL Plus ECL XHyperfilm ECL, Hyperfilm MP ECL Mini-Camera/CCD

12 2.2 ECL Advance SDS-PAGE minive minive6 SDS-PAGEHybond-P Hybond-ECL 2-1 TE 70/TE PVDF100 2 ECL Advance Blocking Agent in PBS-T or TBS-T 1 cm ml 14 PBS-T or TBS-T 2251 PBS-T or TBS-T Tween cm 2 4 ml - 2% ECL Advance Blocking Agent in PBS-T or TBS-T 1 PBS-T or TBS-T ,000100,

13 1 PBS-T or TBS-T ECL Western Blotting Molecular Weight Marker PBS-T or TBS-THRP 15 PBS-T or TBS-T S/NX 10200,000 ECL DualVue Marker HRPS-protein79 HRP100,000500,000 ECL Advance SolutionAECL Advance SolutionB cm ml 28 Hyperfilm ECL % ECL Advance Blocking Agent in PBS-T or TBS- THRP PBS- T TBS-T cmecl Mini-Camera

14 28 PBS-T or TBS-T in PBS-T or TBS-T PBS-T or TBS-T % SDS, 100 mm 2-mercaptoethanol, 62.5 mm Tris-HCl, ph ECL Advance M glycine, ph M guanidine HCl, 50 mm glycine, ph 10.8, 0.05 mm EDTA, 0.1 M KCl, 20 mm 2-mercaptoethanol10

15 2.3 ECL Plus SDS-PAGE minive minive6 SDS-PAGEHybond-P Hybond-ECL2-1, 2-2 TE 70/TE Hybond-PPVDFHybond-ECL Hybond-C extra PVDF100% 5 ECL Blocking agent RPN in PBS-T or TBS-T 1 PBS-T or TBS-T 2251 PBS-T or TBS-T Tween cm ml 3-1 Tween % PBS-T or TBS-T µg/ml 4-1 PBS-T or TBS-T

16 PBS-T or TBS-THRP PBS-T or TBS-T S/NX 1025,000 ECL DualVue Marker HRPS-protein79 PBS-T or TBS-THRP 4060 PBS-T or TBS-T HRPECLHRPRPN1231 1:1,500 SolutionASolutionB401 1 cm ml Hyperfilm ECL PBS- T TBS-T cmecl Mini-Camera

17 28 PBS-T or TBS-T in PBS-T or TBS-T PBS-T or TBS-T102 2% SDS, 100 mm 2-mercaptoethanol, 62.5 mm Tris-HCl, ph M glycine, ph M guanidine HCl, 50 mm glycine, ph 10.8, 0.05 mm EDTA, 0.1 M KCl, 20 mm 2-mercaptoethanol10

18 2.4 ECL SDS-PAGE minive minive8 SDS-PAGEHybond-ECL Hybond-P2-1, 2-2 TE 70/TE Hybond-PPVDFHybond-ECL Hybond-C extra PVDF100% 5 ECL Blocking agent RPN in PBS-T or TBS-T 1 PBS-T or TBS-T 2251 PBS-T or TBS-T Tween cm ml 3-1 Tween % PBS-T or TBS-T 1 PBS-T or TBS-T µg/ml , ,000

19 PBS-T or TBS-THRP PBS-T or TBS-T PBS-T or TBS-THRP 4060 PBS-T or TBS-T S/N X10 10, ECL DualVue Marker HRPS-protein HRPECLHRPRPN1231 1:1, HRPProtein A1,0005,0001,000HRPRPN12311,500HRP RPN10515,000 Detection Reagent1 Detection Reagent cm ml 1 Hyperfilm ECL PBS- T TBS-T cmecl Mini-Camera

20 28 PBS-T or TBS-T in PBS-T or TBS-T % SDS, 100 mm 2-mercaptoethanol, 62.5 mm Tris-HCl, ph PBS-T or TBS-T M glycine, ph M guanidine HCl, 50 mm glycine, ph 10.8, 0.05 mm EDTA, 0.1 M KCl, 20 mm 2-mercaptoethanol10

21 ECL PlusECL Advance 3.4 ECLECL Plus 3.5 DABECL Plus 3.6 ECL Mini-CameraHyperfilm

22 3.1 ECL AdvanceECL PlusECL ECL ECL Advance 11 PVDF100 2 ECL Advance1 3 PBS-TTBS-T2 4 1/10,0001/25,0001/50,0001/75,0001/100, X10 1/200, ECL PlusECL 1 15 µghybond-ppvdfhybond-ecl 1PVDF PBS-0.1 Tween 20PBS-T /1001/5001/1,0001/5,0001/10, ECL Plus 1/25,000ECL1/10, ECL PlusECL

23 /3,0001/5,0001/7,5001/10,0001/25, ECL AdvanceECL PlusECL ECL Advance7 HRP2 2 HRP 2

24 3.2 HRP 22 1 ECL Blocking agentrpn212510%pbs-t TBS-T10 ECL Advance Hybond-PPVDF Hybond-ECL 2 PBS-TTBS-T PBS-TTBS- T PBS-T TBS-T310 4 ECL Mini-CameraX 3.3 ECL PlusECL Advance

25 50 ngbsahybond-ppvdfbsa HRPNA934ECL Plus/ECL Advance XHyperfilm ECL5 ng ng ECL Advance 150, ECL Plus 15, , ,000 ECL AdvanceECL Plus102 ECL PlusECL AdvanceECL Plus24 ECL Plus ngbsasds-pagehybond-ppvdfbsa 1:20,000HRPNA9341:200,000ECL Plus/ECL AdvanceXHyperfilm ECL10 ECL Advance ECL Plus ECL AdvanceECL Plus8 ECL Plus

26 3.4 ECLECL Plus 20 ngbsasds-pagehybond-ppvdfbsa HRPNA934ECL Plus/ECLX Hyperfilm ECL10 A,, ECL B,, ECL Plus X10ECL ECL Plus42.5 ECLECL PlusECL DABECL Plus 2 BSA10 µgsds-pagehybond-ppvdf BSAHRPNA934ECL Plus DAB A,, DAB DAB B,, ECL Plus X Hyperfilm ECL ECL PlusDAB BSA ECL PlusDABS/N ECL Plus2050HRP

27 3.6 ECL Mini-CameraHyperfilm ECL Mini-Camera XCCDECL Mini- Camera 7493 mmecl Mini-Camera HRPNA µlecl PlusECLDABECL PlusECL5DAB 15XHyperfilm ECLECL Mini-CameraISO3000 Mini-Camera Mini-Camera DAB5 ECL ECL ECL Plus ECL Plus Hyperfilm ECL Mini-Camera Hyperfilm ECL Mini-Camera DAB

28 3.7 ECL S/N ECL 3014 ECL Blocking agentrpn2125 ECLECL Plus ECL Advance Tween 20 5%0.1%Tween20PBSTBS ECLECL Plus 5%PBSTBS 1% Fish GelatinRPN416 2%4 BSA PBS0.33%PBS 2%BSA 0.02%10% Protein A

29 NP-40Triton Tween-200.3%0.1%5% 1% ECL blocking agentrpn % BSA 3% Fish GelatinRPN41610% in PBS a b 3-1 SDS-PAGEPY20 absa in TBS-T b in TBS-T

30 3.8 ECL HRP HRP 20 µg/lanehybond-ecl 2 10%PBS-TTween0.11.5PBS-T ,000 PBS1 13,000PBS1 4 5 PBS-T53 HRP IgG-HRPIgM-HRPPBS-T13,00030 IgM-HRPPBS-T13, PBS-0.3%Tween2053 PBS-T53 ECL1Hyperfilm30 PBS-TPBS-0.3%Tween %H 2O 2 in PBS30%H 2O 2PBS1:130HRP PBS 11 PBS-T53 12 ECL1Hyperfilm1 13 PBS-T ,000PBS-T30 15 PBS-T53 16IgG-HRPPBS-T13, PBS-0.3%Tween2053PBS-T53 18 ECL1Hyperfilm1 HRPHRP 15%

31 3-2 Rat Kidney Lysate 20 µg/hybond-ecl RPN202QD 0.1% Tween 20PBS10% 1 -mouse IgG-HRPSigma IgM-HRPCappel mouse IgM-HRP 30 ECL 1Hyperfilm ECLRPN %H 2O 2PBS30ECL 1 Hyperfilm ECL 1 PBS 3-4 1Anti-mouse IgG-HRP 30 PBSECL Hyperfilm ECL30

32 ECL Mini-Camera 4.3 Hyperfilm 4.4 Typhoon CCD 7 8

33 4.1 ECL2 ECL Mini-Camera ECL CCDCCD ECL Mini-Camera Hyperfilm CCD RI

34 4.2 ECL Mini-Camera ECL Mini-Camera... CB-103ECLAlkPhos Direct, Gene Images cm ISO cm cm cm

35

36 4.3 Hyperfilm 20X Hyperfilm20 1X 2X

37 ECLHyperfilm ECL(14) Sensitize RPN ECL 4 CCD 10 µl600 ng450 ng300 ng150 ng60 ng ng2 Hybond-ECL 1:20HRPNA9311:3,000ECL Hyperfilm ECL60 ng UltraScan XL Laser densitometer OD 2 UltraScan XL Laser densitometer 600, 450, 300, 150, 60 ng1, 2 Hyperfilm ECL15 kda)

38 4.4 Typhoon 1 2 Typhoon Typhoon 1 Typhoon TyphoonPCTyphoon ScannerControl 2 scan acquisition modechemiluminescencesetupsensitivityhighpmt voltage950v OKPixel sizesample orientationpress samplefocal planeimage analysis 3 Scan Save 4 ImageQuant 5 Typhoon

39 CCD BSAHybond-P ng BSA1:50,000HRPNA9341:200,000ECL AdvanceCCDImageMaster-CL BSA ngr2 ImageMaster-CL 30,000,000 25,000,000 20,000,000 15,000,000 10,000, ,000 R CCD ng

40 ECL Plus ECL Plus Typhoon ECL Plus Typhoon 6 7 8

41 cm ml ECL PlusDetection Reagent AB4011 cm µlecl Plus5 33 mm (nm) (nm) ECL Plus HRP 2 ECF AP 3 DDAOphosphate AP Horseradish Peroxidase 3 Alkaline Phosphatase

42 5.2 Typhoon 1Typhoon 2 TyphoonECL Plus mm 3Press SampleFocal Plane3 mmpixel Size 100 µmsensitivity Normal PMT V Typhoon (-tubulin) ECL Plus 0.5 ng ECF 24 ng DDAO phosphate 12 ng S/N3 5-1abTyphoon Typhoon5 a) b) 5-1 a ECL Plus ng b ng Typhoon Trio488 nm

43 mini VE 6.2 TE70 / TE MultiProcessor 6 7 8

44 6.1 minive minive mm ml ml 210% TEMEDn- 1 3n- 41 µ mm 1.0 mm 1.5 mm

45 mm 3MINMAX SDS-PAGE1020 ma/ PVDF 6-3a,f b,ecd 4afBlot Module ml 5Blot Module L4 6 7Towbin Buffer ma12 6-3

46 6.2 TE 70 / TE 77 TE 70 / TE TE 70TE mm PVDF 6 a41 b5a c2b d4c kg 150 V ma / cm 2 41Native 2 20 V 10 V

47 1 2 3

48 6.3 MultiProcessor MultiProcessor... ECL ECL ECL Plus ECL Standard Enhanced Clean ECL Plus ECL

49 9 14 DNA DNA 1.0 mm 0.5 mm SDS, Native mm 0.5 mm Immobiline DryPlate SDS, Native 1.5 mm IEF mm CBB SDS, Native 1.0 mm 0.5 mm CBB SDS, Native 0.75 mm CBB SDS, Native 1.5 mm CBB IEF mm Clean

50 ECL ECL ECL 7.1 ECL Western Blotting metallopeptidase 7.2 2D DIGE / ECL Plus Western Blotting

51 Identification of metallopeptidases from germ cell conditioned medium using ph gradient 2-D electrophoresis and ECL immunodetection ECL Western Blotting metallopeptidase ECL metallopeptidase N-arginine dibasic convertaseaminopeptidase Bthimet oligopeptidase metallopeptidase Pineau SD 5 GCCM GCCM 50 µg 400 µl 8 M 0.5 % Triton X % DTT 0.5 %(v/v) Pharmalyte 3-10 Orange G Immobiline DryStrip 3-10 NL 18 cm) MultiTemp III EPS 3500XL Multiphor II 1 SDS-PAGE ECL GCCM N- arginine dibasic (NRD) convertase (narlysin, EC ) aminopeptidase B (ApB, EC ) thimet oligopeptidase (TOP, EC ) Immobiline DryStrip 6M 2 % SDS 10 % 0.5 % DTT 50 mm Tris-HCpH DryStrip 6M 2% SDS 10% 4.5% 50 mm Tris-HCl(pH 6.8) 10 SDS-PAGE ExcelGel SDS Gradient XL ECL Western Blotting Molecular Weight Marker Voltage Current Power Step (V) (ma) (W) Time IEF h h 3 3, h SDS-PAGE 1 1, min 2 1, min 3 1, h 40 min

52 m (kda) ph (non-linear) 7-1GCCM GCCM 50 µgipg DryStrip 3-10NL ExcelGel SDS gradient 12-14% GC metallopeptidaseecl 6 PVDF AuroDye Forte Kit PVDF Novablot 1 cm ma 1 7-2ECLAB GCCMthimet oligopeptidase 7-1 Standard Spot Number 7210N testicular phoshatidylethanolamine binding protein ECL Western Blotting Molecular Weight Marker 97,40068,00046,00031,00020,10014,400 Da ECLWestern blotting ECL SDS-PAGE 1D 3% BSA deglycosylated TBS 50 mm Tris-HCl (ph 7.6) 150 mm NaCl 1 4 TBS deglycosylated 0.1%(v/v) Tween 20 / 1% BSA 0.1%(v/v) Tween 20 / TBS 1 1/8,000 2 HRP 1/3,000 ECL Hyperfilm 7-2ECLmetallopeptidase Standard Spot Number mkda pi thimet oligopeptidase NRD convertase aminopeptidase B

53 1 2 1 thimet oligopeptidase ECL 2 ECL 3 ECL Western Blotting Molecular Weight Marker 7-3 NRD convertase NRD convertasehrp A thimet oligopeptidase NRD convertaseb ECL metallopeptidase 100 mm / 2%(w/v) SDS / 62.5 mm Tris-HCl(pH 6.7) %(v/v) Tween 20 / TBS % BSA deglycosylated TBS 50mM Tris-HCl (ph 7.6) 150 mm NaCl 1 1. Boitani, C. et al., Cell Differentiation 9, (1980). 2. Kramer, J. M. and Erickson, R. P., J. Reprod. Fert. 64, (1982). 3. Jutte, N. H. P. M. et al., J. Exp. Zool. 233, (1985). GCCM metallopeptidase ECL 4. O' Brien, D. A., Biol. Reprod. 37, (1987). 5. Pineau, C. et al., J. Androl. 14, (1993). 6. Heukeshoven, J. and Dernick, R., Electrophoresis 9, (1988).

54 Detection and identification of tyrosine phosphorylated protein 2D DIGE PVDF 2D DIGE MS/MS Ettan DIGE Arabidopsis thaliana 87 MS/MS D DIGE 32 P thaliana 7-5 A. thaliana 2 PY20 1 4G ECL Plus Western Blotting Detection Reagents 130 Mb A. thaliana 4 2D DIGE Ettan DIGE Differential Gel Electrophoresis CyDye Cy2 Cy3 Cy5 CyDye 5 2D DIGE Arabidopsis A. thaliana

55 Dithiothreitol Tris Sodium Dodecylsulfate (SDS) Glycerol TEMED Ammonium Persulfate Agarose Glycine Bind-Silane Multiphor II Electrophoresis System HRP Cy52D DIGE SE 600 Standard Vertical Electrophoresis Unit Dual Gel Caster EPS 3501 XL Power Supply Glass plates, cm, low fluorescence Ettan Spot Picker Reference Markers Ettan DIGE Hybond-P RPN2020F ECL Plus Western Blotting Detection Reagents RPN2132 ECL Phosphorylation module RPN2220 Cy5 mono-reactive Dye Pack PA D DIGE 2D DIGE SYPRO Ruby protein gel stain (Invitrogen) Sequencing Grade Modified Trypsin (Promega) PepMap C18 column (LC Packings) Tween (ICI Americas Inc.) Investigator ProGest (Genomic Solutions) Q-ToF tandem mass spectrometer CapLC capillary chromatograph, MaxEnt 3 (Micromass Ltd.) SE 600 Standard Vertical Electrophoresis Unit SE 600 Ruby CyDye DIGE Fluor Cy2 minimal dye, 25 nmol CyDye DIGE Fluor Cy3 minimal dye, 25 nmol CyDye DIGE Fluor Cy5 minimal dye, 25 nmol Pharmalyte ph Immobiline DryStrip, 18 cm, ph Immobiline DryStrip Reswelling Tray Immobiline DryStrip Cover Fluid Urea CHAPS DIGE A. thaliana µg/ml mm Tris-HCl ph mm EDTA 2 mm DTT 10,000 g 10 80%

56 M urea 2 M thiourea 4% CHAPS 30 mm Tris- HCl ph 8.5 Bradford CyDye DIGE Fluor minimal dye Dimethylformamide Aldrich 1 nmol/µl 5 50 µg 400 pmol 2 µl 7-3 B 6 M urea 2 M thiourea 50 mm Tris ph % glycerol 10% SDS 4.8% iodoacetamide 15 Dual Gel Caster cm 1 mm 12.5% SDS Immobiline DryStrip 192 mm glycine 0.2% [w/v] SDS 25 mm Tris SE 600 Standard Vertical Electrophoresis Unit 1 25 ma ma Cy2 CyDye DIGE Fluor minimal dye Cy3 Cy mm lysine CyDye DIGE Fluor minimal dye 400 pmol 1 µl M urea 2 M thiourea 4% CHAPS 20 mg/ml DTT 4% Pharmalyt ph µg 350 µl Immobiline DryStrip Reswelling Tray Immobiline DryStrip ph4-7, 18 cm 8 Immobiline DryStrip Cover Fluid Multiphor II Electrophoresis System V for 1,000 Vh 2 3,000 Vh gradient from 500 V to 3,500 V 3 70 kvh at 3,500 V A 6 M urea 2 M thiourea 50 mm Tris ph % glycerol 10% SDS 1% DTT Immobiline DryStrip 15 Typhoon 9400 Cy2 488 nm excitation wavelength 520 nm bandpass (BP) 40 nm (520 BP 40) emission filter Cy3 532 nm excitation wavelength 580 BP 30 emission filter Cy5 633 nm excitation wavelength 670 BP 30 emission filter 100 µm 60,000 80,000 PMT ImageQuant Tools version 2.3 Ettan DIGE Analysis Software version 4.00 DeCyder Cy2 Cy3 Cy2 Cy5 DeCyder Differential D DIGE BVA Cy2 CyDye DIGE Fluor minimal dye Cy3 CyDye DIGE Fluor minimal dye Cy5 CyDye DIGE Fluor minimal dye 1 10 min control ARA min treated ARA min control ARA min treated ARA min control ARA 57B 10 min treated ARA 57B 4 10 min control ARA min treated ARA h control ARA 1 24 h treated ARA h control ARA 3 24 h treated ARA h control ARA h treated ARA 65 ARAA. thaliana

57 3,500 3% BSA TBS-T 1,000 HRP PY20 ECL Phosphorylation module 1 ECL Plus Western Blotting Detection Reagents Variation Module landmark BVA DeCyder Biological Cy2 Typhoon 9400 Cy5 ECL Plus ECL Plus 457 nm excitation wavelength 505 nm long-pass filter Cy5 ECL Plus 7-5 Paired Student's t-test p 0.05 proteins of interest 25 mm Tris 200 mm 20% Hybond-P 150 µg 30 V Hoefer TransPhor units 4 monofunctional Cy5 0.1% Tween 20 ICI Americas Inc. PBS 10 mm, ph 7.4 PBS-T 1 PBS-T 100% 10 3 DeCyder ECL Plus protein of interest 7-5 A. thaliana 500 µg Immobiline DryStrip 140 kvh 2D PlusOne Bind-Silane 2 fluorescent reference marker SYPRO Ruby Invitrogen 10% 6% Typhoon 9400 SYPRO Ruby 532 nm excitation wavelength 610 BP 30 emission filter DeCyder 2 x-y Pick List Ettan Spot Picker 2 mm 96 Typhoon 9400 excitation wavelength 670 BP 30 emission filter nm Investigator ProGest Genomic Solutions mm TBS-T 10 mm Tris-HCl ph mm NaCl 0.1% Tween ml 3% BSA 1 4 TBS-T DTT iodoacetamide Promega 25 mm 6.5 ng/µl 10 µl 60

58 mm 5% % Micromass CapLC capillary chromatograph Q-ToF Micromass MS/MS 0.1% 6 µl PepMap C18 column 300 µm 0.5 cm LC Packings 0.1% 3 Cy5 Cy2 Cy3 1 µl/min data acquisition 0.1% 5 70% 20 3,500 V 540 1, Data-dependent acquisition MS/MS collision gas collision voltage V MS/MS MaxEnt 3, Micromass SwissProt/TrEMBL miss cleavages 1 1 initial mass tolerance 50 ppm MS/MS NCBI nr BLAST 13 DeCyder 2D DIGE 3,132 SYPRO Ruby 500 µg 3 3,315 Master Master Master 2D DIGE 7 1, min control/treated h control/treated Paired Student's t-test 10 p p unpaired p min control/treated Paired < h control/treated Paired < min control/24-h control Unpaired < Phosphorylated N/A

59 7-6A Cy3 A Cy5 7-6A 7-6B HRP Cy5-NHS ester PY20 30 mm PY20 HRP B ECL Plus Plus ECL A 7-6. A. 10Cy3Cy5 B. DeCyder BVA A B B 8 3,420 1,799 Cy5 ECL Plus DeCyder BVA 7-8 Cy5 C D DIGE g h i 7-7. A. Cy5 10Cy5 B. ECL PlusA ECL Plus C. D. C 2D DIGE MS/MS 2 f

60 Cy5 ECL Plus 7-8. Cy5 a. Glutamine synthetase cytosolic isozyme b. Serine acetyltransferase c. Thioredoxin reductase 2 d. Glutathione S-transferase ERD13 e. Putative epimerase /dehydratase f. g. 5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase h. Tubulin beta-4 chain i. Adenosine kinase Q-ToF MS/MS ECL Plus DeCyder BVA BVA p 5-methyltetrahydropteroyltriglutamate- 10-min control/treated homocysteine methyltransferase 1.26 Putative thioredoxin (probable protein 24-h control/treated disulfide isomerase 2) 1.20 Tubulin beta-4 chain 10-min control/treated Adenosine kinase 1 10-min control/treated control 10-min/control 24-h Translationally controlled tumor 10-min control/treated protein-like protein 1.28 Heat shock protein 81-2 control 10-min/control 24-h

61 22 5 2D DIGE MS 1. Chivasa, S. et al. Electrophoresis 23, (2002). 2. Ducret, A. et al. Electrophoresis 21, (2000). ECL Plus Western Blotting Detection reagents 3. Leimgruber, R. M. et al. Proteomics 2, (2002). 4. Kaul, S. et al. Nature 408, (2000). 5. Tonge, R. et al. Proteomics 1, (2001). Cy5 Typhoon 9400 Cy5 6. May, M. J. and Leaver, C. J. Plant Physiology 103, (1993). 7. Unlu, M. et al. Electrophoresis 18, (1997). 8. Sanchez, J. C. et al. Electrophoresis 18, (1997). DeCyder DeCyder 2D DIGE Cy5 9. Jeno, P. et al. Anal. Biochem. 224, (1995). 10. Wilm, M. et al. Nature 379, (1996). 11. Shevchenko, A. et al. Anal. Chem. 68, (1996). 12. Wait, R. et al. Electrophoresis 22, (2001). 13. Altschul, S. F. et al. Nucleic Acids Res. 25, (1997). Ettan DIGE More information can be obtained in the Ettan DIGE system section at University of Durham Durham UK Toni Slabas Stephen Chivasa Bongani Ndimba Imperial College of Medicine London UK Robin Wait

62 ECL ECL

63 ) 1.1.1) ( ) (Stokes' radius) (Rainbow 82) ECL K (15) 4 SDS NP %SDS SDS

64 1.1.2) (>150 kda) 1.1.3) (<15 kda) Hybond- PPVDF 1.2) 1.3) 1.2) 1.3.1) 1.1) 1.3.2) 1.4) 1.4.1) 1.4.2) 1.4.3) Tween 1.4.4) 1.4.5) ECLECL Advance (0.5 ml)1 21 µlhrp ECL PlusSolutionA 1 ml SolutionB 25 µl

65 1.5) 1.5.1) 1.5.2) HRP 1.5.3) 1.6) 1.6.1) 1.6.2) HRP 2 2.1) 1) 2.1) 1) 2.2) 2.2) 2.3) 2.3.1) (1msec) Pre-flash Unit (RPN2051) Wratten6B 540 nm ) (12) 2.3.3) HRP 3 3.1) 3.1)

66 3.2) 3.2) 3.3) 3.3) (3 ) 3.4) 3.4) 4 4.1) 4.1) 1 4.2) 4.2.1) 4.2.2) 4.3) 4.3) ECL 4.4) 4.4) 4.5) 4.5) 0.2 µm 5 5.1) 5.1) / (3 ) ECL -

67 5.2) 5.3) 5.4) 5.2) 5.3) 5.4.1) ECL Advance 2%0.2 ml/cm ) 5.4.3) 5.4.4) (10%) 5.4.5) Tween ) Tween ) 5.4.8) ) 110% 0.53% 1%(PVP) TBS-TPBS-T (15-23) 5.5) 5.5.1)

68 5.5.2) Hybond-PHybond-ECL 5.5.3) 5.5.4) ECL 5.5.5) 5.6) 5.6.1) 5.6.2) Tween 20 Tween ) 5.7.1) ) 5.8) 5.8.1) 15 (3 ) 5.8.2) 5 10 ( )

69 6) 6.1) 6.1) 6.2) 6.2) SDS-PAGE 6.3) 6.3)

70 HRP C 6 -tubulin 12% SDS-PAGEHybond-ECL-tubulin (1:1,000)Ig(RPN10011:500)HRP (RPN1051)ECL15Hyperfilm ECL C 6 -tubulin 12% SDS-PAGEHybond-ECL-tubulin 1:1,000HRPIg(NA9311:1,000) ECL Hyperfilm ECL PBS-T ECL Hyperfilm ECL

71 D 5 mm DTT11% SDS- PAGE () 10 V/cm20 1% PBSD IgG-ECL D D mg/ml D. mg/ml cellshela1% SDS15 ng 12% SDS-PAGEP10 V/cm2 5% TBS1/300 IgG:30 µg/mligg:30 µg/ml1tbs-t 1041TBS-T104ECL 3 HRPIgG ( -), HRPIgG ( -), ()

72

73 1. µ µ µ µ APSTEMED PlusOne PlusOne PlusOne PlusOne PlusOne

74 PlusOne D.W. PlusOne PlusOne PlusOne -. gdtt SDS ml-- PlusOne PlusOne PlusOne PlusOne SDS ml- PlusOne PlusOne PlusOne ph

75 2. Towbin 25 mm Tris ph8.3, 192 mm, 20% v/v PlusOne PlusOne PlusOne ph SDS.w/v 3. PBSPhosphate buffered salineph7.580 mm Na 2HPO 4, 20 mm NaH 2PO 4, 100 mm NaCl TBSTris-bufferd salineph7.620 mm Tris, 137 mm NaCl PBS-TPBS-Tween TBS-TTBS-Tween ECL Advance 2824

76 ECL PlusECL ECL DualVue Western Blotting Marker ECL DualVue Western Blotting Marker3 7S-tagged S-Protein-HRPS-tagged ECLECL Plus 1 ECL DualVue Western blotting markers1530 SDS µl10% -1SDS 5 µl-sds 10 µl 3 Hybond-ECLHybond-PPVDF 4 HRPS-protein-HRP conjugateecl 130ECL DualVue MarkerS-tagS-ProteinS-tagged ECL S-ProteinS-tag Richards,F.M and Wyckoff, H.W. (1971) in The Enzymes, Vol. IV (Boyer, P.D. Ed ) p , Academic Press, New York. Kim,J. S. and Raines, R.T. (1993 ) Protein Sci. 2, ECL 12.5

77 1S-Protein HRP conjugatex Hybond-ECL/ Hybond-P/PVDF ECL 1/5000 1/10,000 ECL Plus 1/10,000 1/20,000 ECL Advance 1/100,000 1/200,000 Mr A B Mr

78 4.2. ECL Western Blotting Molecular Weight Markers 11 µl µl 10 µltowbin(25)30v 15 4 Hybond-ECL ECL PlusECLHRP HRP RPN1231, 1:1,500 ECL AdvanceHRP HRP 1:100,000500, ECL HRP ECL Advance Blocking AgentPBS-0.1 Tween20ECL Advance Blocking AgentECL (26) HRP ECL

79 µl µl % VHybond-ECL V HRP (RPN :)ECL Hyperfilm ECL ECL. kda Phosphorylase b() (rabbit muscle) Bovine serum albumin() Ovalbumin() (hen egg white) Carbonic anhydrase() (human erythrocyte) Trypsin lnhibitor() (soybean) Lysozyme() (hen egg white) Electrophoretic mobility (Rf) Long molecular weight Trailing Edge Leading Edge 4.3 Full-Range Rainbow Molecular Weight Markers kda CBB µl - LMW -- HMW SDS --

80 4.4 High-Range/Low-Range Rainbow Molecular Weight Markers - CBB µl -

81 5 5.CBB 0.1% CBB30%10% 30% 10% 5.2

82 References 1) ISACSSON, U. and WATERMARK, G., Anal. Chim. Acta, 68, pp , ) WHITEHEAD, T. P., et al., Clin. Chem., 25, pp , ) ROSEWELL, D.F. and WHITE, E.H., Methods in Enzymology,. 57, DELUCA, M.A.(Ed). Academic Press, New York, pp , ) MOTSENBOCKER, M.A., J.Biolum. Chemilum., 2, pp.9-16, ) AKHAVEN-TAFTI, H. et. al., Clin. Chem., 41, pp , ) AKHAVEN-TAFTI, H. et. al., Biolum. And Chemilum. Fundamentals and Applied Aspects, pp , Chichester, ) ELIOR, PELES. and SALAH, S. BACUS., Cell., 69, pp , ) XIN-YUAN, FU., Cell., 70, pp , ) PAUL, L. STEIN. and HON-MAN, LEE., Cell., 70, pp , ) KATHLEEN, A. SIEMERS. and RACHEL, WILSON., Science, 260, pp , ) T, MEIER. and S, ARNI., Anal. Biochem., 204, pp , ) KAUFMANN, S. H. et al., Anal. Biochem., 161, pp.89-95, ) RUFF-JAMISON, S, et. al., Science, 261, pp , ) LIFE SCIENCE., 7, pp.12-13, Amersham International plc, ) JOHNSTONE, A. and THORPE, R., Immunochemistry in Practice., Blackwell Science Publications, ) GERSHONI, J. M. and PALADE, G. E. Anal. Biochem., 131, pp.1-15, ) TOWBIN, H. and GORDON, J., J. Immunol. Meth., 72, pp , ) PELUSO, R. W. and ROSENBERG, G. E., Anal. Biochem., 162,pp , ) GERSHONI, J. M. and PALADE, G. E., Anal. Biochem.,124, pp , ) BURNETTE, W. M., Anal. Biochem., 112, pp , ) BITTNER, M., KUPFERER, P. and MORRIS, C. F., Anal. Biochem., 102. pp , ) LIN, W. and KASAMATSU, H., Anal. Biochem.,128, pp , ) ANDREWS, A.T., Electrophoresis: Theory, Thechniques and Biochemical and Clinical Application, Second Edition in Monographs on Physical Biochemistry, edited by A. R. Peacock and W. R. Harrington, Oxford Science Publication, ) LEAMMILI, U.K., Nature., 227, pp , ) TOWBIN, H.STAEHELIN, T. and GORDON, J., Proc. Natl. Acad. Sci (USA), 76, pp , ) HOFFMAN, W.L. and JUMP, A.A., Anal. Biochem., 181, pp , 1989.

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