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

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Presentation on theme: "Separation of proteins in the 1 st dimension Dr Kevin Mills Institute of Child Health, UCL, London."— Presentation transcript:

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

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

3 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

4 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

5 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

6 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

7 Separation of proteins in the 2 nd dimension

8 2D-PAGE 2 nd dimension: separation according to mass Polyacrylamide gel Dr Kevin Mills Institute of Child Health, UCL, London

9 2D-PAGE 2 nd dimension: separation according to mass Polyacrylamide gel Molecular Sieve Dr Kevin Mills Institute of Child Health, UCL, London

10 2D-PAGE 2 nd dimension: separation according to mass Polyacrylamide gel + ve

11 Content on this website is the copyright of the Great Ormond Street Hospital for Children and the Institute of Child Health unless otherwise stated. Where copyright applies, visitors can download material for private research, study or in-house use only. Visitors must not copy, distribute, or publish any material from this website. Any other use of copyright material requires the formal permission of the Great Ormond Street Hospital for Children and the Institute of Child Health. Information specifically for patients and families has been reviewed for accuracy, but we hold no responsibility for alterations made thereafter without our consent. The content of this presentation is the copyright of Dr Kevin Mills, UCL Institute of Child Health. Visitors must not copy, distribute, or publish any material from this presentation. It is strictly forbidden to alter this presentation in any way. Any other use of this presentation requires formal permission from Dr Mills. (c) Dr Kevin Mills, UCL Institute of Child Health, London, 2006.


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