1 Principle of 2-D Electrophoresis 1. First dimension: denaturing isoelectric focusing separation according to the isoelectric point 2. Second dimension: SDS electrophoresis separation according to the molecular weight 2-D electrophoresis resolves a few thousand protein spots

2 29-Apr-04 2-D electrophoresis: traditional method

3 Add Sample to 1 st Dimension Strips and Focus

4 April 28, Placing the Ettan ™ SDS gel into the cassette

5 April 28, Place equilibrated IPG strip onto 2 nd Dimension acidic end gel surface up

6 Load and seal the IPG strip onto the gel surface

7 Insert Cassette into Ettan Dalt

8 Theoretical pI and M r map of yeast cell proteins (calculated from MIPS data) Theoretical pI Mr / kDa From: Wildgruber et al. Electrophoresis. 21 (2000)

9 Wide pH Gradient: 3 – 11 NL Mouse liver extract IPG 24 cm, pH 3-11NL From A. Görg Proteomics Department Technische Universität Munich pH 3 pH 11

10 10 Why don`t we see this pattern? Post translational modifications Not all proteins are expressed Regulatory proteins are expressed in low copy numbers Missing proteins: –hydrophobic –high molecular weight –very basic Proteome is not static!

11 The Dynamic Range of Expression: Avogadro´s Challenge Copies / cell , ,000 Fluor dye(1 ng) 20 mg 2 mg 200 µg 20 µg 2 µg Coomassie (100 ng) 2,000 mg 200 mg 20 mg 2 mg 200 µg

12 2-D Electrophoresis of Mouse Liver Proteins pH 4 pH 9 kDa Görg et al. Electrophoresis 16 (1996)

13 April 28, Wide pH gradient: 3 – 11 NL Mouse liver extract IPG 24 cm, pH 3-11 From A. Görg Proteomics Department Technische Universität Munich pH 3 pH 11

14  1491  1564  218  1429 Mouse liver proteins From A. Görg et al. (1999) IPG IPG IPG IPG 5,5 - 6,7 Number of spots

15 IPG strips with overlapping pIs pH pH 3-11 pH pH pH

16 Increased Resolution: Blow - Ups of Spots IPG 4-7 IPG 5-6 IPG 4-7 IPG 5,5-6,7 Mouse liver proteins From A. Görg et al. (1999)

17 Two-Dimensional Gel Based Proteomics Low pIHigh pI 1st Dimension IEF Disease Tissue High Mass Low Mass 2nd Dimension SDS-PAGE Healthy Tissue

18 2-D Electrophoresis - Strengths Physico-chemical parameters of proteins measured Non-destructive separation of intact proteins Isoforms and post-translational modifications displayed Multiplexing, DIGE Quantitative method, internal standard (DIGE) High resolution, particularly after pre-fractionation High throughput, parallel runs Multiple detection, blotting, applicable Efficient fraction collector

19 April 28, High Protein Loads Problem: Highly Abundant Proteins

20 April 28, Highly Abundant Proteins Standard Strip Holder Manifold

21 April 28, Staining of IPG Strips (cont. urea, detergent) Acid Violet 17 Staining: (Patestos NP et al. Electrophoresis. 9 (1988) ) fix for 20 min in 20% TCA, wash for 1 min in 3% phosphoric acid, stain for 10 min in 0.1 % Acid Violet 17 solution in 10% phosphoric acid, destain 3  in 3% phosphoric acid until background is clear, wash 3  1 min with H 2 O dist, impregnate with 5 % glycerol, air dry.

22 April 28, Casting SDS gels – important points HQ reagents: PlusOne labelled chemicals are a benchmark TEMED not too old Freshly made APS Precool monomer solution mix (containing the TEMED) Add APS short before use Pour solutions quickly in one go

23 April 28, Ettan TM Dalt II Gel on film support 1 mg E. coli strain B IPGphor 24 cm pH ETTAN TM Dalt gel 12.5 % T Colloidal CBB G250 staining

24 BACS-SDS gels Synaptic Membrane Preparation

25 Blue Native PAGE for Membrane Protein Complexes Add Coomassie dye into cathodal tank Dye competes with nonionic detergents Negatively charged proteins (like SDS) No aggregation Soluble in detergent-free solution Proteins migrate as blue bands Schägger, H.& von Jagow, G. (1991). Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical Biochemistry 199(2),

kDa 440 kDa 232 kDa 140 kDa 67 kDa MarkerEtioplast Chloroplast 2D Blue Native-PAGE/SDS-PAGE Dr. L. Eichacker, Botanik, LMU München Native Blue Electrophoresis Separation of complexes with PAGE in presence of Coomassie Brilliant Blue (no SDS) Schägger H. In: Attardi GM, Chomyn A, Eds. Methods in Enzymology 264 (1996) Werhahn W, Braun H-P. Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis. Electrophoresis 23 (2002) First Dimension

27 2-D Blue Native/SDS-PAGE Second Dimension

28 Two-Dimensional Blue Native/SDS PAGE