1D and 2D Protein Electrophoresis - Institut de Cancérologie et d ...

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Multiplexing – CyDye Fluors 488 nm Cy2 520 BP 40 532 nm Cy3 Cy5 580 BP 30 633 nm 500 600 670 BP 30 Minimal cross-talk between the fluors because the dyes are spectrally well resolved Typhoon is using single wavelength light to excite fluors
The power of relating to house-keeping protein • Loading the exact same amount of protein is uncertain • Strip and re-probe from an ECL blot : new uncertainties Loss of target proteins unevenly across the blot Detect unspecific signal by poor stripping especially for proteins of the same size • Relating phosho-protein to total (non-phospho and phospho) protein : It can be valuable to also relate to an unrelated house keeping protein, since you can not be sure that your total protein level is not varied in your experiment. Use of ECL Plex eliminates all these uncertainties
Page 1 and 2: - temperature + + + + + + + + + + +
Page 3 and 4: SDS Polyacrylamide Gel Electrophore
Page 5 and 6: Practical hints Always use Tris bas
Page 7 and 8: Disc electrophoresis in readymade g
Page 9 and 10: Pre-cast PhastGel run in 30 minutes
Page 11 and 12: SDS electrophoresis SDS is an anion
Page 13 and 14: Blotting and Specific Detection
Page 15 and 16: 100ug Gel staining versus blotting
Page 17 and 18: Chemiluminescent Detection - ECL Pr
Page 19 and 20: Protein bound to membrane blocked w
Page 21 and 22: ECL Plex - relative quantitative We
Page 23 and 24: GE Healthcare Life Sciences Imaging
Page 25: CyDye TM Properties Very bright •
Page 29 and 30: Detection of proteins from rat brai
Page 31 and 32: Mr. Delaive Biochimie Cellulaire de
Page 33 and 34: Blue Native PAGE For the analysis o
Page 35 and 36: Nonionic detergents for solibilizat
Page 37 and 38: Steps after solubilization Incubati
Page 39 and 40: Buffer system for BN PAGE Blue load
Page 41 and 42: Blue Native- PAGE of Protein Comple
Page 43 and 44: 2D Blue Native/SDS-Electrophoresis
Page 45 and 46: Resolution of mitochondrial superco
Page 47 and 48: 8 th Blue Native Electrophoresis Co
Page 49 and 50: Cationic Detergent PAGE For the ana
Page 51 and 52: Cationic detergent acidic gel PAGE
Page 53 and 54: DIGE: 16-BAC / SDS PAGE gels for hy
Page 55 and 56: Isoelectric Focusing Separation acc
Page 57 and 58: IEF with carrier ampholytes Pharmal
Page 59 and 60: Immobilized pH Gradients (IPG) Acry
Page 61 and 62: Two-dimensional Electrophoresis For
Page 63 and 64: Sample preparation Avoiding protein
Page 65 and 66: SDS in 2-D sample preparation Best
Page 67 and 68: Protein precipitation Clean-up from
Page 69 and 70: -Rat tongue tissue (26ug) -pH 3-10,
Page 71 and 72: Resolubilization possibilities •P
Page 73 and 74: The Trap platform Scale down from m
Page 75 and 76: Protein Enrichment products • NHS
Page 77 and 78:
Make your own protein enrichment to
Page 79 and 80:
Repeatability study: enrichment of
Page 81 and 82:
Isoelectric Focusing Separation acc
Page 83 and 84:
Principle of 2-D electrophoresis 1.
Page 85 and 86:
Sample loading on pre-rehydrated IP
Page 87 and 88:
IPGphor run recording graphs
Page 89 and 90:
Streaking in basic pH region DTT an
Page 91 and 92:
Micropreparative focusing with DeSt
Page 93 and 94:
Basic gels: pH 7-11 NL New propriet
Page 95 and 96:
SDS PAGE chambers for Proteomics 25
Page 97 and 98:
Casting SDS gels and overlay gel ed
Page 99 and 100:
Wide pH gradient: 3 - 11 NL Mouse l
Page 102 and 103:
Protein Detection Methods Coomassie
Page 104 and 105:
Deep Purple Staining - fluorescent
Page 106 and 107:
Deep Purple - applications 2D gel 2
Page 108 and 109:
Carbamylation trains Impurities in
Page 110 and 111:
Salt in the gel Cell washing with P
Page 112 and 113:
Equilibration with SDS buffer 15 mi
Page 114 and 115:
2D Electrophoresis GE Healthcare Li
Page 116:
Amersham Biosciences AB, a General
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