protein biology & & WESTERN WORKFLOW
Contents 2 Separate NuPAGE 3 NuPAGE 4 Bolt & PowerEase 5 6 7 Transfer Bolt 8 2 9 Detect 10 11 MY 12 13 3.0 14
3 www.lifetechnologies.com/western Separate 180 2 Invitrogen Bolt Invitrogen NuPAGE Bis-Tris ph Thermo Scientific Pierce Power Stainer 6 11 Transfer Bolt Thermo Scientific Pierce Power Blotter 10 300kDa PVDF 5 10 Invitrogen iblot 2 7 Detect Invitrogen ibind Western System 2.5 HRP ECL ECL Plus Thermo Scientific MYECL Imager CCDUV 2
Separate www.lifetechnologies.com/separate Bolt www.lifetechnologies.com/proteingels NuPAGE NuPAGE ph NuPAGE NuPAGE 2 150kDa NuPAGE ph A NuPAGE B - 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 35 12 NuPAGE - A NuPAGE 4 12 B 4 20 Lanes1, 6, 7, 10 Mark12Lane2 46 Lane3 Lanes 4,5Lane 8 Lane 9 2 8%, 10%, 12%, 4 12% 1.5 200 kda 12 35 50 7%, 3 8% 30 400 kda 8 60 150kDa 3
NuPAGE NuPAGE 88813 2 XCell4 4 26 100 NuPAGE XCell SureLock Mini-Cell EI0001 2 NuPAGE XCell4 SureLock Midi-Cell WR0100 4 1 100 NuPAGE NuPAGE 2 MES MOPS1 2 MES MOPS NuPAGE & * NuPAGE LC5800 LC5801 ** NuPAGE LC5688 www.lifetechnologies.com/nupage 10% 20% 30% 40% 50% % of length of gel 60% 70% 80% 8% 10% 4 12% 12% 3 8% 7% MES MOPS MES MOPS MES MOPS MES MOPS TA TA 260 kda 260 kda 160 kda 260 kda 260 kda 160 kda 260 kda 260 kda 110 kda 500 kda 260 kda 160 kda 290 kda 80 kda 110 kda 160 kda 500 kda 240 kda 260 kda 110 kda 160 kda 60 kda 80 kda 160 kda 80 kda 110 kda 110 kda 160 kda 50 kda 60 kda 160 kda 60 kda 40 kda 290 kda 80 kda 110 kda 80 kda 160 kda 50 kda 50 kda 240 kda 110 kda 116 kda 80 kda 60 kda 30 kda 40 kda 60 kda 40 kda 97 kda 50 kda 110 kda 80 kda 60 kda 20 kda 50 kda 160 kda 50 kda 30 kda 40 kda 80 kda 60 kda 15 kda 30 kda 66 kda 30 kda 116 kda 40 kda 40 kda 20 kda 50 kda 10 kda 97 kda 60 kda 30 kda 15 kda 20 kda 40 kda 55 kda 50 kda 66 kda 20 kda 15 kda 20 kda 10 kda 30 kda 3.5 kda 30 kda 10 kda 55 kda 15 kda 40 kda 40 kda 15 kda 10 kda 20 kda 40 kda 20 kda 10 kda 90% 30 kda 3.5 kda 3.5 kda 15 kda 3.5 kda 20 kda 15 kda 10 kda 100% 10 kda NuPAGE NuPAGE NuPAGE 4% 3 8% NuPAGE +TA Buffer * 6% 3 8% NuPAGE +TA Buffer 8% 7% NuPAGE +TA Buffer 10% 10% NuPAGE +MOPS Buffer 12% 10% NuPAGE +MOPS Buffer 14% 12% NuPAGE +MOPS Buffer 16% 12% NuPAGE +MES Buffer 18% 12% NuPAGE +MES Buffer NuPAGE 4 12% 3 8% NuPAGE +TA Buffer 4 12% NuPAGE +MOPS Buffer 4 20% 4 12% NuPAGE +MES Buffer 8 16% 4 12% NuPAGE +MOPS Buffer 10 20% 12% NuPAGE +MOPS Buffer 10% 10% NuPAGE +MES Buffer 16% 4 12% NuPAGE +MES Buffer 12% NuPAGE +MES Buffer 10 20% 4 12% NuPAGE +MES Buffer 4
Bolt & Bolt NuPAGE 3 ph Bolt NuPAGE Blot 2 1 Bolt 40 μl 10 well 16 A Bolt B TX 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 B 2. Bolt 20μL Bolt B TX Lane 1 6 Lanes 2 4, 6 7: 250 Lane 5 46 Lane 8: p53 Lane 9: GST Lane 10 PowerEase Invitrogen PowerEase 90W Power Supply PS0090 PowerEase 90W Power Supply www.lifetechnologies.com/bolt PowerEase 300W Power Supply PS0300 PowerEase 300W Power Supply 10 10 4 www.lifetechnologies.com/powerease 5
www.lifetechnologies.com/proteinladders Protein Ladders 26632 8 Low 3.4 100 kda 26614 14 10 200 kda LC0725 8 20 1,200 kda 26616 10 Low 10 170 kda 26619 9 10 250 kda LC5699 9 30 460 kda 26634 10 10 260 kda 26625 8 40 300 kda LC5602 9 Western 20 220 kda 26635 10 11 250 kda LC5928 7 11 155 kda LC5606 10 10 160 kda IEF Marker 3 10 3921201 13 3 10 pi kda 460 268 238 171 117 71 55 41 31 3 12% 4 16% PageRuler Low Range 26632 PageRuler Unstained 26614 NativeMark Unstained Protein Standard LC0725 NativePAGE Bis-Tris Gels Gel load volume of 5 μl Stained using coomassie R-250 PageRuler Prestained 26616 PageRuler Plus Prestained 26619 HiMark Pre-stained LC5699 NuPAGE 3 8% Tris-acetate SDS buffer kda 220 120 100 80 60 50 40 30 20 Visual detection Infrared detection SimplyBlue stained InVision stained Spectra Multicolor Broad Range 26634 Spectra Multicolor High Range 26625 MagicMark XP LC5602 NuPAGE Bis-Tris Gel, blotted to nitrocellulose, detected w/westernbreeze Chemiluminescent Kit PageRuler Prestained NIR 26635 BenchMark Fluorescent LC5928 BenchMark His-tagged LC5606 IEF Marker 3-10 39212-01 Novex ph 3 10 Gel 6
Pierce Power Stainer 22833 10 1 2 1 LCD Pierce Power Stainer Pierce Power Stainer 11 230 * 45 10 0.25 250 30 5 www.lifetechnologies.com/powerstainer Protein Stains Coomassie 25 0.5 0.5 Asp Glu 24620 24612 S6653 Better LC6060 Better LC6100 Better S6650 Best Best 24615 24600 S12001 www.lifetechnologies.com/proteinstains 7
Transfer PVDF 5 10 7 www.lifetechnologies.com/transfer 1 Bolt Bolt Bolt 2 Bolt 2 Bolt 2 60 www.lifetechnologies.com/miniblotmodule Bolt B IKK HCK EPHB3 MAPK14 FLT1 DDR2A Bolt Plus B B 50 GST GST Bolt 4 12 B 4 20 Bolt Bolt B PVDF GST 1 Bolt B1000 A B Pierce Power Blotter 22834 10 300kDa 1 5 10 10 300kDa 7.08.44 8.0 13.52 Low (<25kDa) Thermo Scientific Precise Protein Gel (Tris-glycine) Nitrocellulose Cyclophilin B (21kDa) Mid (25-150kDa) NuPAGE 4-12% Bis-Tris Gel Nitrocellulose PLK-1 (67kDa) High (>150kDa) NuPAGE 4-12% Bis-Tris Gel PVDF mtor (289kDa) Pierce Power Blotter 10 B 10 60 www.lifetechnologies.com/powerblotter Pierce Power Blotter 8
iblot 2 IB21001 7 7 NC A B C PVDF D E F A431 201.2 4 12% Bolt A,D NuPAGE B,EC,F 2 PVDF HRP www.lifetechnologies.com/iblot 9
AP HRP 3,500 1,000 ibind Western System www.lifetechnologies.com/detect ibind Western System SLF1000S 2 SLF 2.5 www.lifetechnologies.com/ibind 40,000 www.lifetechnologies.com/antibodies ibind A431 2 PVDF 1068 1H12 1 1,000 2μl 10μl H L 1 3605.55μL 1 1,800 5.55μL C D A B 800CW 1 3,000 1 15,000 A C 80 680 790 WesternDot 585 625 655 800 1 2 3 4 5 6 EGFR B D Phospho-EGFR Merged www.lifetechnologies.com/westerndot 10
5 2 HRP5 Pierce ECL Substrate 32106 SuperSignal West Pico Chemiluminescent Substrate 32132 Pierce ECL Plus Substrate 34080 SuperSignal West Dura Extended Duration Substrate 34076 SuperSignal West Femto Substrate 34094 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 ECL ECL ECL Plus HRP 20 1 0.5 250 60 30 2 6 8 5 24 8 ECL CCD 4 4 4 www.lifetechnologies.com/chemisubstrates BSA PBS TBS PVDF X 3 10 11
MYECL Imager 62236 MY MY CCDX 2 10 UV MYECL Imager 5 A B CCD X 2 1 A B HeLa 10 MY 3 3 www.lifetechnologies.com/myeclimager myimageanalysis Software MYECL Imager 2.0 myimageanalysis Software v2.0 TIFF JPEG PNG 12
13 Unconjugated Biotin HRP AlkPhos Anti-Human IgA chain affinity purified Goat A18783 A18785 A18781 A18784 Anti-Human IgA chain cross-adsorbed Goat A18789 A18791 A18787 A18790 Anti-Human IgE chain affinity purified Goat A18795 A18797 A18793 A18796 Anti-Human IgE chain cross-adsorbed Goat A18801 A18803 A18799 A18802 Anti-Human IgG H+L affinity purified Goat A18807 A18809 A18805 A18808 Anti-Human IgG Fc affinity purified Goat A18819 A18821 A18817 A18820 Anti-Human IgG Fc highly cross-adsorbed Goat A18831 A18833 A18829 A18832 Anti-Human IgG Fc cross-adsorbed Goat A18825 A18827 A18823 A18826 Anti-Human IgG H+L cross-adsorbed Goat A18813 A18815 A18811 A18814 Anti-Human IgG/A/M affinity purified Goat A18849 A18851 A18847 A18850 Anti-Human IgM chain affinity purified Goat A18837 A18839 A18835 A18838 Anti-Human IgM chain cross-adsorbed Goat A18843 A18845 A18841 A18844 Anti-Human KappaLight Chain affinity purified Goat A18855 A18857 A18853 A18856 Anti-Human KappaLight Chain cross-adsorbed Goat A18861 A18863 A18859 A18862 Anti-Human IgG H+L affinity purified Rabbit A18905 A18907 A18903 A18906 Anti-Human IgM chain affinity purified Rabbit A18911 A18913 A18909 A18912 Anti-Mouse IgG H+L affinity purified Chicken A15977 A15979 A15975 A15978 Anti-Mouse IgG H+L cross-adsorbed Chicken A15983 A15985 A15981 A15984 Anti-Mouse IgG H+L affinity purified Donkey A16013 A16015 A16011 A16014 Anti-Mouse IgG H+L highly cross-adsorbed Donkey A16019 A16021 A16017 A16020 Anti-Mouse IgG H+L affinity purified Goat A16068 A16070 A16066 A16069 Anti-Mouse IgG H+L cross-adsorbed Goat A16074 A16076 A16072 A16075 Anti-Mouse IgG H+L highly cross-adsorbed Goat A16080 A16082 A16078 A16081 Anti-Mouse IgG Fc affinity purified Goat A16086 A16088 A16084 A16087 Anti-Mouse IgG Fc cross-adsorbed Goat A16092 A16094 A16090 A16093 Anti-Mouse IgG H+L affinity purified Rabbit A16162 A16164 A16160 A16163 Anti-Mouse IgG H+L cross-adsorbed Rabbit A16168 A16170 A16166 A16169 Anti-Rat IgG H+L affinity purified Chicken A18729 A18731 A18727 A18730 Anti-Rat IgG H+L cross-adsorbed Chicken A18735 A18737 A18733 A18736 Anti-Rat IgG H+L affinity purified Donkey A18741 A18743 A18739 A18742 Anti-Rat IgG H+L highly cross-adsorbed Donkey A18747 A18749 A18745 A18748 Anti-Rat IgG H+L affinity purified Goat A18867 A18869 A18865 A18868 Anti-Rat IgG H+L cross-adsorbed Goat A18873 A18875 A18871 A18874 Anti-Rat IgG H+L affinity purified Rabbit A18917 A18919 A18915 A18918 Anti-Rat IgG H+L cross-adsorbed Rabbit A18923 A18925 A18921 A18924 Anti-Rabbit IgG H+L affinity purified Chicken A15989 A15991 A15987 A15990 Anti-Rabbit IgG H+L cross-adsorbed Chicken A15995 A15997 A15993 A15996 Anti-Rabbit IgG H+L affinity purified Donkey A16025 A16027 A16023 A16026 Anti-Rabbit IgG H+L cross-adsorbed Donkey A16031 A16033 A16029 A16032 Anti-Rabbit IgG H+L highly cross-adsorbed Donkey A16037 A16039 A16035 A16038 Anti-Rabbit IgG H+L affinity purified Goat A16098 A16100 A16096 A16099 Anti-Rabbit IgG H+L cross-adsorbed Goat A16106 A16108 A16104 A16107 Anti-Rabbit IgG H+L highly cross-adsorbed Goat A16112 A16114 A16110 A16113 Anti-Rabbit IgG Fc affinity purified Goat A16119 A16122 A16116 A16120 Anti-Rabbit IgG Fc cross-adsorbed Goat A16126 A16128 A16124 A16127 Anti-Rabbit IgG H+L affinity purified Sheep A16174 A16176 A16172 A16175 Anti-Goat IgG H+L affinity purified Chicken A15965 A15967 A15963 A15966 Anti-Goat IgG H+L cross-adsorbed Chicken A15971 A15973 A15969 A15972 Anti-Goat IgG H+L affinity purified Donkey A16001 A16003 A15999 A16002 Anti-Goat IgG H+L cross-adsorbed Donkey A16007 A16009 A16005 A16008 Anti-Goat IgG H+L affinity purified Rabbit A16138 A16140 A16136 A16139 Anti-Goat IgG H+L cross-adsorbed Rabbit A16144 A16146 A16142 A16145 Anti-Goat IgG Fc cross-adsorbed Rabbit A16150 A16152 A16148 A16151 Anti-Bovine IgG H+L affinity purified Goat A18753 A18755 A18751 A18754 Anti-Cat IgG H+L affinity purified Goat A18759 A18761 A18757 A18760 Anti-Cat IgG H+L affinity purified Rabbit A18879 A18881 A18877 A18880 Anti-Chicken IgY H+L affinity purified Goat A16056 A16058 A16054 A16057 Anti-Chicken IgY H+L affinity purified Rabbit A16132 A16134 A16130 A16133 Anti-Dog IgG H+L affinity purified Goat A18765 A18767 A18763 A18766 Anti-Dog IgG H+L affinity purified Goat A18765 A18767 A18763 A18766 Anti-Guinea Pig IgG H+L affinity purified Goat A18771 A18773 A18769 A18772 Anti-Guinea Pig IgG H+L highly cross-adsorbed Goat A18777 A18775 A18778 Anti-Guinea Pig IgG H+L affinity purified Rabbit A18885 A18887 A18883 A18886 Anti-Hamster IgG H+L affinity purified Rabbit A18891 A18893 A18889 A18892 Anti-Hamster IgG H+L cross-adsorbed Rabbit A18897 A18899 A18895 A18898 Anti-Llama IgG H+L affinity purified Goat A16062 A16064 A16060 A16063 Anti-Llama IgG H+L affinity purified Rabbit A16156 A16158 A16154 A16157 Anti-Sheep IgG H+L affinity purified Donkey A16043 A16045 A16041 A16044 Anti-Sheep IgG H+L highly cross-adsorbed Donkey A16050 A16052 A16047 A16051
Qubit 3.0 Fluorometer Q33216 3.0 1μL 20μL DNA RNA W13.6 D25.0 H5.5 3 Qubit Protein Assay 200 L Qubit Qubit 3.0 Fluorometer www.lifetechnologies.com/qubit 14
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