Proteome Analysis July 1, 2009 Learning objectives. Understand the theoretical basis of 2D-SDS Gel Electrophoresis. Understand the latest technique for.

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Presentation transcript:

Proteome Analysis July 1, 2009 Learning objectives. Understand the theoretical basis of 2D-SDS Gel Electrophoresis. Understand the latest technique for identifying proteins using mass spectrometry. Become familiar with the SWISS-2DPAGE Database Workshop on identifying proteins with the pI, MW and a few peptide masses.

What is the proteome? genome transcriptome proteome metabolome

2D-SDS Gel Electrophoresis What is it? This is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right angles to one another. Proteins separate over a large area, having a greater chance of resolution.

First dimension Separation by isoelectric point Isoelectric point-pH at which the net charge on the molecule is zero

Visuals /Tut_2D.html#Section%203

Figure Alberts et al. Molecular Biology of the Cell, 3rd edn.

2nd Dimension-to separate by size Five steps: 1. Isolate proteins 2. Denature proteins 3. Load onto gel 4. Apply voltage gradient 5. Stain

/courses/Molbio/SDSPAGE/SDSPAGE.html

/courses/Molbio/SDSPAGE/SDSPAGE.html

/courses/Molbio/SDSPAGE/SDSPAGE.html

Figure Alberts et al. Molecular Biology of the Cell, 3rd edn.

Figure Lodish et al. Molecular Cell Biology.

Databases of 2D-Gels

manual/manualwork2html02testp9.htm

Laser Detector m/z Ab % Vacuum Plate Linear Mode Reflection Mode

Protein Prospector Another Web-based program is Protein Prospector (UCSF)