Separation of proteins in the 1 st dimension Dr Kevin Mills Institute of Child Health, UCL, London.

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Presentation transcript:

Separation of proteins in the 1 st dimension Dr Kevin Mills Institute of Child Health, UCL, London

+ + Charged electrode - Charged electrode Movement of charged molecules in an electric field Dr Kevin Mills Institute of Child Health, UCL, London

2D-DIGE Labelling of each sample with a fluorescent tag Label with Fluorescent dye (wavelength 1) Label with Fluorescent dye (wavelength 2) Label with Fluorescent dye (wavelength 3) Sample 1Sample 2Sample 3 Mix samples Dr Kevin Mills Institute of Child Health, UCL, London

Isoelectric focusing strip - IEF High pH Low pH 2D-DIGE 1 st Dimension isoelectric focusing of proteins Dr Kevin Mills Institute of Child Health, UCL, London

Isoelectric focusing strip - IEF High pH Low pH 8 hours 2D-DIGE 1 st Dimension isoelectric focusing of proteins Dr Kevin Mills Institute of Child Health, UCL, London

Isoelectric focusing strip - IEF High pH Low pH 16 hours Proteins will migrate in an electric current until their pI = pH and their net charge is zero – isoelectric point 2D-DIGE 1 st Dimension isoelectric focusing of proteins

Separation of proteins in the 2 nd dimension

2D-PAGE 2 nd dimension: separation according to mass Polyacrylamide gel Dr Kevin Mills Institute of Child Health, UCL, London

2D-PAGE 2 nd dimension: separation according to mass Polyacrylamide gel Molecular Sieve Dr Kevin Mills Institute of Child Health, UCL, London

2D-PAGE 2 nd dimension: separation according to mass Polyacrylamide gel + ve

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